Nad synthetase inhibitors and uses thereof

ABSTRACT

Disclosed are compounds that inhibit the microbial NAD synthetase enzyme. For example, disclosed are compounds of the formula Ar 1 —X—Ar 2 —Y—L—Z—Q, wherein Q is Q 1 Ar 3  or Ar 3 Q 1 ; Ar 1 , Ar 2 , and Ar 3  are independently aryl or heteroaryl, optionally substituted with one or more substituents; X, Y, and Z are independently selected from the group consisting of a covalent bond or groups containing one or more of C, H, N, O, S atoms; L is a linker and Q 1  is an alkylenyl, alkylenyl carbonyloxy alkyl, or alkylenyl carbonylamino alkyl group, optionally having a substituent; a covalent bond; a group containing amidine or guanidine function wherein the amidine or guanidine may be optionally N-substituted with an alkyl; or a zwitterion; or a pharmaceutically acceptable salt thereof. Also disclosed are methods which involve the use of the compounds of the present invention, for example, in treating or preventing a microbial infection in a mammal or plant, killing a prokaryote or decreasing prokaryotic growth, disinfecting a material or environment contaminated by a microbe, increasing food animal production, controlling harm to plants by a pest or insect, and combating agroterrorism. Examples of microbes affected by the compounds of the present invention are bacteria and fungi.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of co-pendingU.S. patent application Ser. No. 09/617,258, filed Jul. 14, 2000, whichis a continuation of International Application No. PCT/US99/14839, filedJun. 30, 1999, which in turn is a continuation-in-part of InternationalApplication No. PCT/US99/00810, filed Jan. 14, 1999, and claims thebenefit of U.S. provisional patent application No. 60/097,880, filedAug. 25, 1998 and 60/071,399, filed Jan. 14, 1998. The presentapplication is also a continuation-in-part of co-pending U.S. patentapplication Ser. No. 09/606,256, filed Jun. 29, 2000, which claims thebenefit of U.S. provisional patent application No. 60/141,436, filedJun. 29, 1999, and a continuation-in-part of PCT/US00/18029, filed Jun.29, 2000. The present application is also a continuation-in-part ofInternational Application No. PCT/US01/22203, filed Jul. 13, 2001, whichclaims the benefit of U.S. provisional patent application No.60/218,405, filed Jul. 14, 2000. The disclosures of all of the relatedapplications mentioned herein are incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] Some research that contributed to the invention herein wassupported, in part, by a grant from the Government of the United Statesof America, Defense Advanced Research Projects Agency. The Governmentmay have certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention in general relates to antimicrobial agents,and in particular, to inhibitors of the nicotinamide adeninedinucleotide (NAD) synthetase enzyme of microbes such as bacteria andfungi. The present invention also relates to the various uses of theseantimicrobial agents, including in a method of treating or preventing amicrobial infection in a mammal, in a method of treating the environmentagainst microbial contamination, in agriculture, e.g., in raisingfoodcrops and food animals, in medicine, e.g., to disinfect, sterilize,or decontaminate equipment, devices, rooms, and/or people, and incombating bioterrorism, e.g., agroterrorism.

BACKGROUND OF THE INVENTION

[0004] Drug-resistant infectious bacteria, that is, bacteria that arenot killed or inhibited by existing antibacterial and antimicrobialcompounds, have become an alarmingly serious worldwide health problem.Rubenstein, Science, 264, 360 (1994). It is believed that a number ofbacterial infections may soon be untreatable unless alternative drugtreatments are identified.

[0005] Antimicrobial or antibacterial resistance has been recognizedsince the introduction of penicillin nearly 50 years ago. At that time,penicillin-resistant infections caused by Staphylococcus aureus rapidlyappeared. Today, hospitals worldwide are facing challenges from therapid emergence and dissemination of microbes resistant to one or moreantimicrobial and antibacterial agents commonly in use today. Severalstrains of antibiotic-resistant bacteria are now emerging and arebecoming a threat to human and animal populations, including thosesummarized below:

[0006] Strains of Staphylococcus aureus resistant to methicillin andother antibiotics are endemic in hospitals. Infection withmethicillin-resistant S. aureus (MRSA) strains may also be increasing innon-hospital settings. Vancomycin is the only effective treatment forMRSA infections. A particularly troubling observation is that S. aureusstrains with reduced susceptibility to vancomycin have emerged recentlyin Japan and the United States. The emergence of vancomycin-resistantstrains would present a serious problem for physicians and patients.

[0007] Increasing reliance on vancomycin has led to the emergence ofvancomycin-resistant enterococci (VRE), bacteria that infect wounds, theurinary tract and other sites. Until 1989, such resistance had not beenreported in U.S. hospitals. By 1993, however, more than 10 percent ofhospital-acquired enterococci infections reported to the Centers forDisease Control (“CDC”) were resistant.

[0008]Streptococcus pneumoniae causes thousands of cases of meningitisand pneumonia, as well as 7 million cases of ear infection in the UnitedStates each year. Currently, about 30 percent of S. pneumoniae isolatesare resistant to penicillin, the primary drug used to treat thisinfection. Many penicillin-resistant strains are also resistant to otherantimicrobial or antibacterial drugs.

[0009] Strains of multi-drug resistant tuberculosis (MDR-TB) haveemerged over the last decade and pose a particular threat to peopleinfected with HIV. Drug-resistant strains are as contagious as thosethat are susceptible to drugs. MDR-TB is more difficult and vastly moreexpensive to treat, and patients may remain infectious longer due toinadequate treatment. Multi-drug resistant strains of Mycobacteriumtuberculosis have also emerged in several countries, including the U.S.

[0010] Diarrheal diseases cause almost 3 million deaths a year, mostlyin developing countries, where resistant strains of highly pathogenicbacteria such as Shigella dysenteriae, Campylobacter, Vibrio cholerae,Escherichia coli and Salmonella are emerging. Furthermore, recentoutbreaks of Salmonella food poisoning have occurred in the UnitedStates. A potentially dangerous “superbug” known as Salmonellatyphimurium, resistant to ampicillin, sulfa, streptomycin, tetracyclineand chloramphenicol, has caused illness in Europe, Canada and the UnitedStates.

[0011] In addition to its adverse effect on public health, antimicrobialresistance contributes to higher health care costs. Treating antibioticresistant infections often requires the use of more expensive or moretoxic drugs and can result in longer hospital stays for infectedpatients. The Institute of Medicine, a part of the National Academy ofSciences, has estimated that the annual cost of treating antibioticresistant infections in the United States may be as high as $30 billion.

[0012] In addition, the use of antibiotics in food animal feeds and theextent to which such use contributes to the development of drugresistance have been under recent discussion, see, e.g., C. Marwick,“Animal Feed Antibiotic Use Raises Drug Resistance Fear,” Journal of theAmerican Medical Association, 282(2):120-2, Jul. 14, 1999, and T. R.Shryock, “Relationship between usage of antibiotics in food-producinganimals and the appearance of antibiotic resistant bacteria,”International Journal of Antimicrobial Agents, 12(4):275-8, August 1999.The use of antibiotics as well as biocides can lead to antibiotic ordrug-resistant organisms, see, e.g., A. D. Russel, “Mechanisms ofbacterial resistance to antibiotics and biocides,” Progress in MedicinalChemistry, 35:133-97, 1998.

[0013] Further, spore-forming bacteria can be lethal. For example,Bacillus anthracis causes the deadly disease, anthrax. There exists anuncertainty relating to the efficacy of currently available vaccinesagainst B. anthracis. Further, there is a likelihood that terroristscould employ antibiotic-resistant strains, e.g., engineered strains thatare not recognized by B. anthracis antibodies or common bacteriaengineered to carry the virulence gene (see, e.g., T. C. Dixon et al.,“Anthrax,” New England Journal of Medicine, 341 (11), 815-826, September1999). The foregoing shows that there exists a need for a noveltreatment against spore-forming bacteria, particularly B. anthracis orbacteria carrying the virulence gene of B. anthracis.

[0014] Further, the incidence of serious fungal infections, eithersystemic or topical, continues to increase for plants, animals, andhumans. Fungi are plant-like eukaryotes that grow in colonies of singlecells, called yeasts, or in filamentous multicellular aggregates, calledmolds. While many fungi are common in the environment and not harmful toplants or mammals, some are parasites of terrestrial plants and otherscan produce disease in humans and animals. When present in humans,mycotic (fungal) diseases can include contagious skin and hairinfections, noncontagious systemic infections, and noncontagiousfoodborne toxemias. The incidence of such infections is notinsignificant; in the U.S. approximately 10% of the population suffersfrom contagious skin and hair infections. While few healthy personsdevelop life-threatening systemic fungal infections, immunocompromisedindividuals, such as found in pregnancy, congenital thymic defects, oracquired immune deficiency syndrome (AIDS), can become seriously ill.This is further illustrated by the fact that fungal infections havebecome a major cause of death in organ transplant recipients and cancerpatients.

[0015] Numerous antifungal agents have been developed for topical useagainst nonsystemic fungal infections. However, the treatment ofsystemic fungal infections, particularly in immunocrompromised hosts,continues to be a major objective in infectious disease chemotherapy.The organisms most commonly implicated in systemic infections includeCandida spp., Cryptococcus neoformans, and Aspergillus spp., althoughthere are a number of emerging pathogens. The major classes of systemicdrugs in use currently are the polyenes (e.g., amphotericin B) and theazoles (e.g., fluconazole). While somewhat effective in otherwisehealthy patients, these agents are inadequate in severelyimmunocompromised individuals. Furthermore, drug resistance has become aserious problem, rendering these antifungal agents ineffective in someindividuals.

[0016] One reason for the limited number of systemic antifungal agentsrelates to the fact that, unlike bacteria, which are prokaryotes, yeastand molds are eukaryotes. Thus the biochemical make-up of yeast andmolds more closely resembles eukaryotic human and animal cells. Ingeneral, this has made it difficult to develop antifungal drugs whichselectively target in yeast or mold an essential enzyme or biochemicalpathway that has a close analog in humans and animals.

[0017] In addition, in view of the risks such as toxicity orcarcinogenicity associated with many common pesticides, fungicides, orbactericides, new approaches are needed to control pests, or insects inthe environment, as well as microbial diseases in plants and food crops,see, e.g., D. W. Wong and G. H. Robertson, “Combinatorial chemistry andits applications in agriculture and food,” Advances in ExperimentalMedicine & Biology, 464:91-105, 1999, and S. H. Zahm and M. H. Ward,“Pesticides and childhood cancer,” Environmental Health Perspectives,106, Suppl. 3:893-908, June 1998.

[0018] Bioterrorism, especially agricultural bioterrorism (oragroterrorism), is presently of great concern in this country as well asin many countries throughout the world. See, e.g., Joseph W. Foxell,Jr., “Current Trends in Agroterrorism (Antilivestock, Anticrop, andAntisoil Bioagricultural Terrorism) and Their Potential Impact on FoodSecurity”, in Studies in Conflict & Terrorism, 24, 107-129 (2001); MarkWheelis, “Agricultural Biowarfare and Bioterrorism—An AnalyticalFramework and Recommendations for the Fifth BTWC Review Conference”,14^(th) Workshop of the Pugwash Study Group on the Implementation of theChemical Biological Weapons Conventions, Geneva, Switzerland, November2000; Radford G. David, “Agricultural Bioterrorism—New Frontiers” inBiowarfare, October 2001; Robert P. Kadlec, Chapter 10, BiologicalWeapons for Waging Economic Warfare, Battle of the Future, 21^(st)Century Warfare Issues, Aerospace Power Chronicles; Senator Kay BaileyHutchison, S. 1563, The Agricultural Bioterrorism Countermeasures Act of2001, Senate Floor Speech, Oct. 17, 2001, page S. 10796.

[0019] Given the above, there exists a need to develop novelantimicrobial agents, especially those which act by different mechanismsthan those agents in use currently. There exists a need to developantibacterial agents that preferentially attack microorganisms and killor deactivate the harmful organism without causing any attendantundesirable side effects in a human or animal patient.

[0020] There also exists a need for methods of treating or preventingmicrobial infection, methods for treating an environment, methods fortreating food crops and animals, methods for decontaminating objects,and/or developing countermeasures against bioterrorism, particularlyagrobioterrorism.

[0021] The advantages of the present invention as well as inventivefeatures will be apparent from the description below.

BRIEF SUMMARY OF THE INVENTION

[0022] The present invention ameliorates some of the disadvantages ofpreviously known antimicrobial agents. The present invention providesantimicrobial agents comprising two aryl moieties linked by a suitablelinker, and the antimicrobial agents inhibit the NAD synthetase enzymeof a microbe.

[0023] In accordance with an embodiment, the present invention providesa compound of the formula (I):

Ar₁—X—Ar₂—Y—L—Z—Q  (I)

[0024] wherein Q is Q₁Ar₃ or Ar₃Q₁;

[0025] Ar₁, Ar₂, and Ar₃ are independently aryl or heteroaryl,optionally substituted with one or more substituents; X, Y, and Z areindependently selected from the group consisting of a covalent bond orgroups containing one or more of C, H, N, O, S atoms; L is a linker and

[0026] Q₁ is an alkylenyl, alkylenyl carbonyloxy alkyl, or alkylenylcarbonylamino alkyl group, optionally having a substituent; a covalentbond; a group containing amidine or guanidine function wherein theamidine or guanidine may be optionally N-substituted with an alkyl; or azwitterion;

[0027] or a pharmaceutically acceptable salt thereof.

[0028] The present invention further provides a compound of the formulaA—B—(CH₂)_(n)—O—CO—CH₂—Ph (NMe₃)⁺I⁻, wherein A is a phenyl or indole,optionally substituted with a benzyloxy group; B is a covalent bond oroxygen atom; n is 1-15; and I⁻ is a pharmaceutically acceptable anion.

[0029] Further, the invention provides a method of treating orpreventing a microbial infection in a mammal comprising administering tothe mammal a treatment effective or treatment preventive amount of amicrobial NAD synthetase enzyme inhibitor compound. Still further, amethod is provided of killing a prokaryote with an amount of prokaryoticNAD synthetase enzyme inhibitor to reduce or eliminate the production ofNAD whereby the prokaryote is killed. Moreover, a method is provided ofdecreasing prokaryotic growth, comprising contacting the prokaryote withan amount of prokaryotic NAD synthetase enzyme inhibitor effective toreduce or eliminate the production of NAD whereby prokaryotic growth isdecreased. Further provided is a disinfecting composition comprising amicrobial NAD synthetase enzyme inhibitor. Still further, the inventionprovides a method of disinfecting a material contaminated by a microbe,comprising contacting a contaminated material with a microbial NADsynthetase enzyme inhibitor compound in an amount sufficient to kill ordeactivate the microbe. The present invention provides a method fortreating or preventing a microbial infection in a mammal comprisingadministering to the mammal an effective amount of a compound thatinhibits the enzymatic activity of the microbial NAD synthetase.

[0030] The present invention, in an embodiment, is based in part on thediscovery that NAD synthetase inhibitors are highly effective ininhibiting the growth of a fungus such as yeast, yet exhibit onlymoderate toxicity in animals. Thus, the present invention includes theuse of NAD synthetase inhibitors as antifungal agents for preventing orcontrolling fungal infections such as parasitic yeast and moldinfections in plants, and for the prophylactic or therapeutic treatment,topically and systemically, of fungal infections in humans and animals.The present invention provides a method of killing a fungus with anamount of NAD synthetase enzyme inhibitor to reduce or eliminate theproduction of NAD whereby the fungus is killed. The present inventionalso provides a method of decreasing fungus growth, comprisingcontacting the yeast with an amount of a NAD synthetase enzyme inhibitoreffective to reduce or eliminate the production of NAD whereby fungusgrowth is decreased.

[0031] The present invention also provides a method for increasingproduction of food animals comprising administering to the food animalan effective amount of at least one inhibitor of NAD synthetase of amicrobe capable of infecting the food animal. The present inventionfurther provides a method for the treatment or prevention of infectionby a spore-forming bacterium in an animal comprising treating anenvironment of the animal with an effective amount of at least oneinhibitor of NAD synthetase of the spore-forming bacterium.

[0032] The present invention further provides a method for killing thevegetative cell of a spore-forming bacterium in an environmentcomprising treating the environment with an effective amount of at leastone inhibitor of NAD synthetase of the bacterium.

[0033] The present invention also provides a method for treating afungal or bacterial disease in a plant comprising treating the plant orthe environment of the plant with an effective amount of at least oneinhibitor of NAD synthetase of the fungus or bacterium. The presentinvention further provides a method for treating or preventing harm to aplant due to a pest comprising contacting the plant, or an environmentthereof, with a pesticidal effective amount of a NAD synthetase enzymeinhibitor of the pest.

[0034] The present invention further provides a pharmaceuticalcomposition comprising a compound as described above and apharmaceutically acceptable carrier. The present invention furtherprovides a method for treating or preventing a microbial infection in amammal comprising administering to said mammal an effective amount of acompound that binds to the interface of the NAD synthetase enzyme dimerof the microbe.

[0035] The present invention further provides a method for combatingagroterrorism involving an infective agent on an object comprisingtreating the object with an amount of a compound effective to inhibitthe NAD synthetase of the infective agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1 depicts a reaction scheme wherein the NAD synthetase enzymecatalyzes the final step in the biosynthesis of NAD.

[0037]FIG. 2 schematically illustrates catalytic sites on a bacterialNAD synthetase enzyme.

[0038]FIG. 3 schematically illustrates the blocking of catalytic sitesof a bacterial NAD synthetase enzyme.

SPECIFIC DESCRIPTION OF THE INVENTION

[0039] The present invention provides a microbial NAD synthetase enzymeinhibitor, having the formula I:

[0040] wherein n is an integer of from 1 to 12, R₁-R₇ each,independently, is H, an unsubstituted or a substituted cyclic oraliphatic group, a branched or unbranched group, wherein the linker is acyclic or aliphatic, branched or an unbranched alkyl, alkenyl, or analkynyl group and wherein the linker may also contain heteroatoms.

[0041] R₁-R₇ may also be one of the following groups: H, alkyl, alkenyl,alkynyl, or an aryl. R₁-R₇, may further be a hydroxyl, ketone, nitro,amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen ora common derivatives of these groups. Note that n may also be an integerof from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to9. The “aryl,” moieties may be the same or different.

[0042] As an example, the present invention provides a microbial NADsynthetase enzyme inhibitor, having formula 2:

[0043] wherein X is a C, N, O or S within a monocyclic or bicyclicmoiety, A and B represent the respective sites of attachment for thelinker, n is an integer of from 1 to 12, R₁-R₇ each, independently, isan H, an unsubstituted or a substituted cyclic group, or an aliphaticgroup, or a branched or an unbranched group, wherein the linker is asaturated or unsaturated cyclic group or an aliphatic branched orunbranched alkyl, alkenyl or alkynyl group, and wherein the linker mayalso contain heteroatoms.

[0044] R₁-R₇ may also be one of the following groups: H, alkyl, alkenyl,alkynyl, or an aryl group. R₁-R₇ may also be a hydroxyl, ketone, nitro,amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen orthe common derivatives of these groups. One of skill in the art wouldknow what moieties are considered to constitute derivatives of thesegroups. In further embodiments, n may also be an integer of from 3 to10, more preferable 5 to 9 and, still more preferably 6 to 9.

[0045] In an embodiments, the linker has the formula A-(C,Heteroatom)n-B. For example, the linker may be an amide, ester, ether,or combinations thereof.

[0046] The present invention, in an embodiment, provides a compound offormula (I):

Ar₁—X—Ar₂—Y—L—Z—Q  (I)

[0047] wherein Q is Q₁Ar₃ or Ar₃Q₁;

[0048] Ar₁, Ar₂, and Ar₃ are independently aryl or heteroaryl,optionally substituted with one or more substituents selected from thegroup consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, halo, amino, C₁-C₆ alkylamino,C₁-C₆ dialkylamino, C₁-C₆ trialkylamino, C₁-C₆ alkylamino C₁-C₆ alkyl,C₁-C₆ dialkylamino C₁-C₆ alkyl, C₁-C₆ trialkylamino C₁-C₆ alkyl, azido,amine oxide, hydroxy, carboxyl, C₁-C₆ alkylcarbonyl, C₁-C₆ alkylcarbonylC₁-C₆ alkyl, C₁-C₆ alkylcarbonyloxy, C₁-C₆ alkylcarbonyloxy C₁-C₆ alkyl,C₁-C₆ alkyloxycarbonyl C₁-C₆ alkyl, C₁-C₆ alkyloxycarbonyl, C₁-C₆alkylthio, nitro, nitrosyl, cyano, hydroxylamino, sulfonamido, C₁-C₆dialkyl sulfonamido, C₁-C₆ alkylcarbonylamino, formyl, formylamino,mercaptyl, and heterocyclyl; optionally, a ring nitrogen atom ofheteroaryl Ar₁, Ar₂, or Ar₃ may be quaternized;

[0049] X, Y, and Z are independently selected from the group consistingof a covalent bond, (CH₂)_(m)O, O(CH₂)_(m), (CH₂O)_(m), (OCH₂)_(m),(CH₂CH₂O)_(m), (OCH₂CH₂)_(m), C(═O)O, OC(═O), OC(═O)O, (CH₂)_(m)S,S(CH₂)_(m), (CH₂S)_(m), (SCH₂)_(m), NH, NR, ^(+NR) ₂, C(═O)NH, C(═O)NR,NHC(═O), NRC(═O), CH(OH), and CH(OR), wherein R is C₁-C₆ alkyl and m is0-5;

[0050] L is {(CR₁R₂)_(q)—(W)_(t)—(CR₃R₄)_(r)}_(p), wherein R₁—R₄ areindependently H, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, halo, amino, C₁-C₆ alkylamino,C₁-C₆ dialkylamino, azido, hydroxy, aldehyde, C₁-C₆ acetal, C₁-C₆ ketal,C₁-C₆ alkylcarbonyl, C₁-C₆ alkylcarbonyl C₁-C₆ alkyl, C₁-C₆alkylcarbonyloxy, C₁-C₆ alkylcarbonyloxy C₁-C₆ alkyl, C₁-C₆ alkylthio,nitro, nitrosyl, cyano, sulfonamido, C₁-C₆ alkylcarbonylamino, orheterocyclyl; W is a moiety selected from the group consisting ofalicyclic ring, aromatic ring, heterocyclic ring, combinations ofalicyclic, heterocyclic, and/or aromatic rings, C₂-C₆ alkenyl, dienyl,C₂-C₆ alkynyl, C₁-C₆ alkoxy, C₂-C₆ alkenyloxy, C₂-C₆ alkynyloxy,anhydrido, enol, ketene, amino, imino, hydrazinyl, epoxy, episulfide,amido, amine oxide, urea, urethane, ester, thioester, carbonate,carbonyl, thiocarbonyl, sulfonyl, diazo, sulfonamido, ether oxygen,ether sulfur, thionyl, silyl, peroxide, lactam, lactone, phenylene,monosaccharide, dri-, tri-, and higher polysaccharides, nucleic acid,amino acid, phosphonyl, phosphoryl, and combinations thereof; q, r, andt are independently 0-20; q, r, and t are not simultaneously 0; and p is1-6; L, optionally, further including O, N, or S; and

[0051] Q₁ is (i) a C₁-C₆ alkylenyl, C₁-C₆ alkylenyl carbonyloxy C₁-C₆alkyl, or C₁-C₆ alkylenyl carbonylamino C₁-C₆ alkyl group, optionallyhaving a substituent selected from the group consisting of amino, C₁-C₆alkylamino, C₁-C₆ haloalkylamino, C₁-C₆ haloalkyl C₁-C₆ alkyl amino,C₁-C₆ hydroxyalkylamino, C₁-C₆ hydroxyalkyl C₁-C₆ alkylamino, C₁-C₆dialkylamino, C₁-C₆ trialkylamino, and heterocyclic containing anitrogen atom which may be optionally quaternized, (ii) a C₂-C₆alkylenyl; (iii) methylenyl with the proviso that Z is other thancovalent bond or O(C═O) when Q is Q₁Ar₃ wherein Ar₃ is a phenyl parasubstituted with amino, methylamino, dimethylamino, or trimethylamino orAr₃ is a pyridyl or N-methylpyridyl; (iv) a covalent bond with theproviso that when Ar₃ is pyridyl, N-methyl pyridyl, or phenyl parasubstituted with trimethylaminomethyl group, Z is other than a covalentbond or O(C═O); (v) a group containing amidine or guanidine functionwherein the amidine or guanidine may be optionally N-substituted with aC₁-C₆ alkyl; or (vi) a zwitterion;

[0052] or a pharmaceutically acceptable salt thereof.

[0053] The aryl of Ar₁, Ar₂, and Ar₃ includes 1-3 aromatic rings, forexample, phenyl, naphthyl, or anthracenyl, preferably phenyl. Theheteroaryl of Ar₁, Ar₂, and Ar₃ include 1-3 rings, one or more of whichinclude O, N, or S, preferably N. Examples of heteroaryls includeindole, benzopyranone, benzoxazole, benzothiazole, In embodiments of thecompound of the present invention, Ar₁ is phenyl or phenyl substitutedwith one or more substituents selected from the group consisting ofC₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxy C₁-C₆ alkyl, halo, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino,C₁-C₆ trialkylamino, C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆ dialkylaminoC₁-C₆ alkyl, C₁-C₆ trialkylamino C₁-C₆ alkyl, azido, amine oxide,hydroxy, carboxyl, C₁-C₆ alkylcarbonyl, C₁-C₆ alkylcarbonyl C₁-C₆ alkyl,C₁-C₆ alkylcarbonyloxy, C₁-C₆ alkylcarbonyloxy C₁-C₆ alkyl, C₁-C₆alkyloxycarbonyl C₁-C₆ alkyl, C₁-C₆ alkyloxycarbonyl, C₁-C₆ alkylthio,nitro, nitrosyl, cyano, hydroxylamino, sulfonamido, C₁-C₆ dialkylsulfonamido, C₁-C₆ alkylcarbonylamino, formyl, formylamino, mercaptyl,and heterocyclyl.

[0054] In preferred embodiments of the compounds of the presentinvention, Ar₁ is phenyl or phenyl substituted with one or moresubstituents selected from the group consisting of C₁-C₆ alkoxy, halo,amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, azido, C₁-C₆alkylcarbonyloxy, C₁-C₆ alkylthio, nitro, cyano, sulfonamido, C₁-C₆dialkyl sulfonamido, C₁-C₆ alkylcarbonylamino, and heterocyclyl.

[0055] Embodiments of the compounds of the present invention includecompounds wherein Ar₁ is phenyl, phenyl substituted with one or moreC₁-C₆ alkoxy, particularly phenyl substituted with one or more methoxyor propoxy. Embodiments of the compounds of the present invention alsoinclude compounds wherein Ar₁ is phenyl substituted with one or morehalo, particularly, one, two, or three chloro or fluoro. Embodiments ofthe compounds of the present invention also include compounds whereinAr₁ is phenyl substituted with one or more C₁-C₆ dialkylamino,particularly N,N-dimethylamino. Embodiments of the compounds of thepresent invention further include compounds wherein Ar₁ is phenylsubstituted with one or more azido, nitro, and cyano. Embodiments of thecompounds of the present invention also include compounds wherein Ar₁ isphenyl substituted with one or more C₁-C₆ dialkyl sulfonamido,particularly N,N-dimethyl sulfonamido. Embodiments of the compounds ofthe present invention also include compounds wherein Ar₁ is phenylsubstituted with one or more C₁-C₆ alkylcarbonyloxy, particularlyacetoxy. Embodiments of the compounds of the present invention alsoinclude compounds wherein Ar₁ is phenyl substituted with one or moreC₁-C₆ alkylcarbonylamino, particularly acetylamino. Embodiments of thecompounds of the present invention also include compounds wherein Ar₁ isphenyl substituted with one or more C₁-C₆ alkylthio, particularlymethylthio. Embodiments of the compounds of the present invention alsoinclude compounds wherein Ar₁ is phenyl substituted with one or moreheterocyclyl, particularly diazolyl.

[0056] In accordance with the present invention, embodiments includecompounds wherein Ar₂ is phenyl, optionally substituted with one or moresubstituents selected from the group consisting of C₁-C₆ alkyl, C₁-C₆alkoxy, and C₁-C₆ alkyloxycarbonyl. In a preferred embodiment, Ar₂ isphenyl.

[0057] In accordance with the present invention, embodiments alsoinclude compounds wherein Ar₂ is indolyl or indolyl substituted with oneor more substituents selected from the group consisting of C₁-C₆ alkyl,C₁-C₆ alkoxy, and C₁-C₆ alkyloxycarbonyl. In a preferred embodiment, Ar₂is indolyl, particularly indolyl substituted with one or more C₁-C₆alkylcarbonyloxy. In another preferred embodiment, Ar₂ isbenzopyranonyl.

[0058] In accordance with the present invention, embodiments includecompounds wherein Ar₃ is phenyl, indolyl, or pyridyl, optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, C₁-C₆dialkylamino, C₁-C₆ trialkylamino, and nitro. In a particularembodiment, Ar₃ is phenyl, optionally substituted with one or more C₁-C₆trialkylamino, preferably N,N,N-trimethylamino. In another embodiment,Ar₃ is indolyl.

[0059] In accordance with an embodiment of the present invention, Q isAr₃Q₁ and Q₁ is C₁-C₆ alkylenyl carbonyloxy C₁-C₆ alkyl, optionallyhaving a C₁-C₆ trialkylamino, for example, Q₁ is trimethylaminoethylenyl carbonyloxy t-butyl.

[0060] In accordance with another embodiment, Q is Q₁Ar₃, wherein Q₁ isC₁-C₆ alkylenyl, optionally having a C₁-C₆ trialkylamino or aheterocyclic containing a quaternized nitrogen atom. Examples of Q₁include methylenyl and trimethylamino ethylenyl, and ethylenyl having aN-alkyl pyrrolidinyl, N-alkyl piperidinyl, orN,N-dialkyl-N-tetrahydropyranyl substituent. In certain embodiments, Q₁is a covalent bond, preferably a single bond, e.g., when Ar₃ isN-methylpyridinyl and Z is NH(C═O) or NR(C═O).

[0061] In a preferred embodiment of the compound of the presentinvention, Z is NH(C═O) or NR(C═O), more preferably NH(C═O).

[0062] In an embodiment of the present invention, Q₁ is a zwitterion,for example, an internal salt of a natural or synthetic amino acid. Inanother embodiment of the present invention, Q₁ is a group containingamidine or guanidine function wherein the amidine or guanidine may beoptionally N-substituted with a C₁-C₆ alkyl.

[0063] In a preferred embodiment of the compounds of the presentinvention, t is 0. In a particularly preferred embodiment, R₁-R₄ are H.In another preferred embodiment, q and r are independently 1-7. In yetanother preferred embodiment, p is 1-4. Still further preferredembodiments include compounds wherein q and r are 1, q and r are 2, andone of q and r is 1 and the other of q and r is 2.

[0064] In an embodiment of the compound of the present invention, X isselected from the group consisting of CH₂O, (C═O)O, and covalent bond.In another embodiment of the compound of the present invention, Y isselected from the group consisting of covalent bond and O. An example ofa covalent bond is a single bond. In yet another embodiment of thepresent invention Z is selected from the group consisting of O(C═O),covalent bond, NH(C═O), NR(C═O), O, NR, and ^(+NR) ₂.

[0065] Specific compounds of the present invention include compoundswherein Ar₁ is phenyl or a phenyl substituted with chloro, fluoro,methylthio, methoxy, isopropoxy, N,N-dimethylamino, azido, nitro,acetoxy, cyano, acetylamino, sulfonamido, or diazolyl; X is CH₂O,(C═O)O, or single bond; Ar₂ is phenyl, indolyl, or benzopyranonyl, eachof the Ar₂ may be substituted with methoxycarbonyl; Y is O, (C═O)O, orsingle bond; L is (CH₂)_(n) wherein n is 7-11; Z is O(C═O), NH(C═O), O,single bond, OCH₂, NCH₃, or N⁺; Q₁ is single bond, CH₂—CH(GU)—CH₂,(GU)CH—CH₂, CH₂CH(N⁺R₅R₆R₇)CH₂, wherein GU is guanidine, R₅, R₆, and R₇are alkyl or heterocyclic or together with the N⁺ forms a heterocyclic;and Ar₃ is phenyl, N-methylpyridinyl, N,N,N-trimethylaminophenyl, ornitrophenyl.

[0066] Specific embodiments include compounds wherein Ar₁ is phenyl, Xis CH₂O, Ar₂ is phenyl or indolyl; Y is single bond or O; L is (CH₂)₇ or(CH₂)₈; Z is O, NH(C═O), O(C═O); Q₁ is single bond, n-propyl, CH₂,CH(NMe₃)CH₂, CH₂—CH(GU)—CH₂, (GU)CH—CH₂; and Ar₃ is phenyl, indolyl,hydroxyphenyl, nitrophenyl, and N,N,N-trimethylaminophenyl, wherein thehydroxy, nitro and N,N,N-trimethylamino groups may be present in the o-,m-, or p- position. Other embodiments include compounds wherein Ar₁ iso-, m-, or p- chlorophenyl; X is CH₂O; Ar₂ is phenyl; Y is O; L is(CH₂)₈; Z is NH(C═O) or O(C═O); Q₁ is CH₂, single bond, CH(NMe₃)CH₂, orCH(N-methylpyrrolidinyl)CH₂, and Ar₃ is phenyl, N-methylpyridinyl, orN,N,N-trimethylaminophenyl. Further embodiments include compoundswherein Ar₁ is dichlorophenyl wherein the chlorine atoms may be in the2,3; 2,4; 2,5; 2,6; 3,4; 3,5; or 3,6-position; X is (C═O)O or CH₂O; Ar₂is phenyl; Y is O; L is (CH₂)₈; Z is NH(C═O); Q₁ is single bond, CH₂,CH(NMe₃)CH₂; and Ar₃ is phenyl, N-methylpyridinyl, orN,N,N-trimethylaminophenyl. Additional embodiments include compoundswherein Ar₁ is trichlorophenyl wherein the chlorine atoms may be presentin the 2,3,4; 2,4,5; 2,5,6; 3,4,5; or 3,5,6 position; X is (C═O)O; Ar₂is phenyl; Y is 0; L is (CH₂)₈; Z is NH(C═O); Q₁ is CH₂, CH(NMe₃)CH₂;and Ar₃ is phenyl or N,N,N-trimethylaminophenyl. Other embodimentsinclude compounds wherein Ar₁ is o-, m-, or p- fluorophenyl; X is(C═O)O; Ar₂ is phenyl; Y is 0; L is (CH₂)₈; Z is NH(C═O); Q₁ isCH(NMe₃)CH₂; and Ar₃ is phenyl. Further embodiments include compoundswherein Ar₁ is difluorophenyl wherein the fluorine atoms may be in the2,3; 2,4; 2,5; 2,6; 3,4; 3,5; or 3,6-position; X is (C═O)O; Ar₂ isphenyl; Y is O; L is (CH₂)₈; Z is NH(C═O); Q₁ is CH(NMe₃)CH₂; and Ar₃ isphenyl. Additional embodiments include compounds wherein Ar₁ istrifluorophenyl wherein the fluorine atoms may be present in the 2,3,4;2,4,5; 2,5,6; 3,4,5; or 3,5,6 position; X is (C═O)O; Ar₂ is phenyl; Y isO; L is (CH₂)s; Z is NH(C═O); Q₁ is single bond, CH₂, or CH(NMe₃)CH₂;and Ar₃ is phenyl or N-methylpyridinyl, or N,N,N-trimethylaminophenyl.Additional embodiments include compounds wherein Ar₁ is methoxy phenylor isopropoxy phenyl, wherein the methoxy or isopropoxy group may bepresent in the o-, m-, or p- position; X is (C═O)O or CH₂O; Ar₂ isphenyl; Y is O; L is (CH₂)₈; Z is NH(C═O) or O(C═O); Q₁ is single bond,CH₂, or CH(NMe₃)CH₂; and Ar₃ is phenyl or N-methylpyridinyl, orN,N,N-trimethylaminophenyl. In the embodiments above Q is preferablyQ₁Ar₃.

[0067] Particular examples of compounds of the present inventioninclude:

[0068] wherein I⁻is a pharmaceutically acceptable anion.

[0069] The compounds described above can have a suitable configurationif an asymmetric center is present. Thus, the compounds may be in R, S,or a mixture of R and S forms. Further, in the compounds describedabove, the amino acids employed may be the natural (L) form or theunnatural (D) form.

[0070] Embodiments of the above compounds of formula (I) include:

[0071] The present invention provides in another embodiment, a compoundof the formula A—B—(CH₂)_(n)—O—CO—CH₂—Ph (NMe₃)⁺I⁻, wherein A is aphenyl or indole, optionally substituted with a benzyloxy group; B is acovalent bond or oxygen atom, and I⁻is a pharmaceutically acceptableanion. For example, A is a phenyl group substituted with benzyloxy,chlorobenzyloxy, or methoxybenzyloxy group. The chloro or methoxy groupcan be in any of ortho, para, or meta positions. In embodiments, thechloro or methoxy group is in the ortho or para position. A furtherexample includes a compound where A is an indole substituted withbenzyloxy.

[0072] Specific examples of the compounds of the above embodiment of thepresent invention include compounds selected from the group consistingof:

[0073] wherein I⁻ is a pharmaceutically acceptable anion.

[0074] In a preferred embodiment, the inhibitor of NAD synthetase hasthe Structure 2′:

[0075] wherein Aryl 1 is indolyl or phenyl; Aryl 2 is phenyl, pyridinyl,indolyl, or quinolinyl; and the linker is —(CH₂)_(n)—,—(CH₂)_(n)—O—C(═O)—, —O(CH₂)_(n)—O—C(═O)—, —(CH₂)_(n)—O—C(═O)CH₂—, or—O(CH₂)_(n)—O—C(═O)CH₂—.

[0076] For example, in Structures 2, 2′, and 4, R₁—R₃ are independentlyselected from the group consisting of H, aryloxy, hydroxyaryl, arylC₁-C₆ alkoxy, C₁-C₆ alkoxy, C₁-C₆ alkoxycarbonyl, C₁-C₆ alkyl, C₁-C₆alkylcarbonyl, arylcarbonyl, nitro, halo, carboxy, halo C₁-C₆ alkyl,perhalo C₁-C₆ alkyl, triphenymethoxy, phenylcarbonylamino, C₁-C₆alkoxycarbonyl C₂-C₆ alkenyl, arylcarbonyl C₂-C₆ alkenyl, benzofuranylcarbonyl, C₁-C₆ alkylbenzylfuranyl carbonyl, arylaminocarbonyl,arylcarbonyloxy, aminocarbonyl, C₁-C₆ alkoxycarbonylamino,phthalidimido, morpholino, pyrrolidinyl, phenylhydantoinyl, andacetylpiperazinyl; and R₆-R₇ are independently selected from the groupconsisting of H, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆trialkylammonium, C₁-C₆ N-alkyl, and C₁-C₆ alkoxycarbonyl. In anembodiment, R₃—R₄ are independently H.

[0077] In some embodiments, Aryl 1 is indolyl. In some otherembodiments, Aryl 1 is phenyl. In certain embodiments, Aryl 2 is phenyl.In certain other embodiments, Aryl 2 is pyridinyl. In furtherembodiments, Aryl 2 is quinolinyl. In other embodiments, Aryl 2 isindolyl.

[0078] In certain embodiments, particularly where Aryl 1 is indolyl orphenyl, more particularly indolyl, R₁-R₃ are independently selected fromthe group consisting of H, phenoxy, hydroxyphenyl, benzyloxy, methoxy,methoxycarbonyl, isopropyl, butyl, acetyl, phenylcarbonyl, nitro,fluoro, carboxy, trifluoromethyl, triphenylmethoxy, phenylcarbonylamino,methoxycarbonyl ethenyl, phenylcarbonyl ethenyl, benzofuranyl carbonyl,butylbenzylfuranyl carbonyl, phenylaminocarbonyl, phenylcarbonyloxy,aminocarbonyl, methoxycarbonylamino, phthalidimido, morpholino,pyrrolidinyl, phenylhydantoinyl, and acetylpiperazinyl.

[0079] In other embodiments, particularly where Aryl 1 is phenyl, R₁-R₃are independently selected from the group consisting of H, phenoxy,hydroxyphenyl, benzyloxy, acetyl, phenylcarbonyl, nitro, phenylcarbonylethenyl, benzofuranyl carbonyl, butylbenzylfuranyl carbonyl,phenylaminocarbonyl, phenylcarbonyloxy, aminocarbonyl, andmethoxycarbonylamino.

[0080] Other examples of inhibitors of NAD synthetase has the Structure300:

[0081] wherein Y is C, N, O, S, ester, amide, or ketone, n is an integerof from 1 to 12, a is an integer from 1-3, and R₁-R₅ each,independently, is H, unsubstituted or substituted cyclic group or analiphatic group, a branched or an unbranched group, or an alkyl,alkenyl, or alkynyl, or an aryl group.

[0082] A further example of the inhibitor of NAD synthetase has theStructure 400:

[0083] wherein Y is C, N, O, S, ester, amide, or ketone; Z is C, N, O,or S; AA is a natural or unnatural stereoisomer of an α-, β, γ, orδ-amino acid in which the carboxyl carbonyl is attached to Z, and theamino grouping may be a primary, secondary, tertiary, or quaternaryammonium compound; n is an integer of from 1 to 12; and R₁-R₅ each,

[0084] independently, is H, unsubstituted or substituted cyclic group oran aliphatic group, a branched or an unbranched group, or an alkyl,alkenyl, alkynyl, aryl, aryl alkyl, or aryl alkoxy group.

[0085] In Structures 300 and 400, R₁-R₂ may also be H, hydroxyl, ketone,nitro, amino, amidino, guanidino, carboxylate, amide, ester, sulfonate,halogen, alkoxy, or aryloxy group.

[0086] Particular examples of inhibitors of NAD synthetase are 5940,5949, 5951, 5409, 5948, 5270, 5939, 5947, 5953, and 5274:

[0087] The present invention further provides a method for treating orpreventing a microbial (e.g., bacterial or fungal) infection in a mammalcomprising administering to said mammal an effective amount of acompound that binds to the dimer interface of the NAD synthetase enzymeof the microbe (bacterium or fungus).

[0088] In the method of killing yeast, as well as in the method ofdecreasing the growth of yeast, the NAD synthetase enzyme inhibitor is acompound that selectively binds with catalytic sites or subsites on ayeast NAD synthetase enzyme to reduce or eliminate the production of NADby the yeast. In such methods, it is particularly preferably that thereis little or no inhibitory activity on the host cell. For example, whenthe method is utilized to inhibit yeast activity in a mammal, it ispreferred that there is little or no attendant affect on the NADsynthetase activity of the host. In one embodiment, the host is amammal. In a further embodiment, the host is a plant.

[0089] In one embodiment, the invention provides administering anantifungal agent to a mammal in need of such treatment or prevention. Inone embodiment, the fungal agent that causes the infection is yeast. Inseparate embodiments of the methods of administering, the antifungalagent comprises one or more compounds disclosed herein.

[0090] Further provided by the invention herein is preferably a methodof killing yeast with an amount of yeast NAD synthetase enzyme inhibitorcompound to reduce or eliminate the production of NAD whereby the yeastis killed. The present invention further provides a method of decreasingyeast growth, comprising contacting the yeast with an amount of yeastNAD synthetase enzyme inhibitor effective to reduce or eliminate theproduction of NAD whereby yeast growth is decreased is also provided.

[0091] The present invention provides, in an embodiment, a method forincreasing production of a food animal comprising administering to thefood animal an effective amount of at least one inhibitor of NADs of amicrobe capable of infecting the food animal.

[0092] In another embodiment, the present invention provides a methodfor the treatment or prevention of infection by a spore-formingbacterium in an animal comprising treating an environment of the animalwith an effective amount of at least one inhibitor of NADs of thespore-forming bacterium. In a further embodiment, the present inventionprovides a method for killing the vegetative cell of a spore-formingbacterium in an environment comprising treating the environment with aneffective amount of at least one inhibitor of NADs of the bacterium. Anexample of a spore-forming bacterium is a biological warfare agent,e.g., Bacillus anthracis.

[0093] In still another embodiment, the present invention provides amethod for treating a fungal or bacterial disease in a plant comprisingtreating the plant or an environment of the plant with an effectiveamount of at least one inhibitor of NADs of the fungus or bacterium. Ina further embodiment, the present invention provides a method for atreating plant comprising the treating the plant, or an environmentthereof, with a pesticidal effective amount of at least one inhibitor ofNADs of a pest. An example of the plant is a food crop.

[0094] In yet another embodiment, the present invention provides amethod for disinfecting, sterilizing, or decontaminating an objectcomprising treating the object with an effective amount of at least oneinhibitor of NADs of a microbe. The microbe is a microorganism, e.g.,bacterium or fungus. An example of a fungus is mold or yeast.

[0095] Any suitable object can be disinfected, sterilized, ordecontaminated. Examples of suitable objects include an article ofclothing, an animal, an organ of an animal, a structure, an equipment, afurniture, an environment, a food crop, a chicken, a chicken skin, andan egg, e.g., egg shell. In accordance with the present invention, theenvironment being disinfected, sterilized, or decontaminated can beland, air, or water, or a combination thereof.

[0096] An example of the environment includes a medical environment.Thus, for example, a medical device, medical equipment, hospital, orsurgical room can be disinfected. Medical personnel also can bedisinfected or decontaminated. In accordance with the present invention,medical devices such as implantable medical devices, e.g., catheters canbe disinfected, sterilized, or decontaminated. Medical equipment such asa surgical equipment may also be disinfected, sterilized, ordecontaminated. Further, the organs of animals, including human, can bedisinfected or decontaminated. An example of an organ is the digestivetract.

[0097] In a further embodiment, the present invention provides a methodfor controlling insect population in an environment comprising treatingthe environment with an effective amount of at least one inhibitor ofNADs of the insect. Any suitable environment can be treated. Forexample, a household environment or an agricultural environment can betreated.

[0098] For the treatment of food animals to increase production, theinhibitor or antimicrobial agent may be mixed with animal feed at atypical concentration of 1-500 mg per kg of feed. Alternatively, similarconcentrations may be added to the animals' drinking water. Furtheralternatively, the antimicrobial agent may be administered as an oralpill or may be injected, either intramuscularly or intravenously.

[0099] The method of the present invention in an embodiment is useful inthe prophylaxis or therapy of biological warfare agents, including, butnot limited to, the spore-forming bacterium such as Bacillus anthracisor a microorganism carrying the virulent gene of a spore-formingbacteria such as Bacillus anthracis. In Bacillus anthracis and otherspore-forming bacteria, NADs is required for outgrowth of the germinatedspore. Since inhibitors of NADs also prevent vegetative growth, thisrepresents two different points of attack on the life cycle of thesebacteria and should provide extremely effective prophylaxis and/ortherapy.

[0100] In the treatment of plants, in a typical application, theantimicrobial agent in a suitable vehicle is sprayed onto growing plantsto either prevent or treat fungal and/or bacterial diseases.Alternatively, application may be made by deposition of solutions orsolid preparations on the soil near growing plants.

[0101] In an application of NADs inhibitors as pesticides forcontrolling pests and insects in the household and/or for agriculturaluses, NADs inhibitors with pesticidal or insecticidal activities and ina suitable vehicle, e.g., organic or aqueous vehicle, are sprayed inareas of homes that are commonly treated with existing insecticidalpreparations. In a typical agricultural application, the pesticidal orinsecticidal agent in a suitable vehicle is sprayed onto growing plantsto either prevent or treat infestation by insects. Alternatively,pesticidal or insecticidal application to plants may be made bydeposition on the soil near growing plants.

[0102] In a typical application for disinfection, sterilization ordecontamination of structural surfaces, a solution of the microbicidalcompound in a suitable vehicle would be painted, sprayed, or soaked (byimmersion into a solution) onto the surface of the object. For treatmentof the soil or ground, a solution of the microbicidal agent in asuitable vehicle may be sprayed onto or soaked into the ground, or asolid form may be mixed with the soil. The microbicidal agent may alsobe added to contaminated water supplies in sufficient concentration(1-100 micromolar) to cause sterilization. In processing, handling, andpackaging animal foods, such as eggs or chickens, a solution of themicrobicidal compound in a suitable vehicle may be painted, sprayed, orsoaked (by immersion into a solution) onto the surface of the food.Numerous related beneficial applications are possible, includingdecontamination of chicken skins, e.g., to reduce Salmonellatyphimurium, egg shells (carriers of Salmonella), and disinfection ofother foods.

[0103] In the field of sterilization, disinfecting and decontaminationincluding, microbicidal concentrations of NADs inhibitors have thepotential for use in a variety of situations benefiting fromsterilization or decontamination, including the treatment of clothing,surfaces of structures, equipment, furniture, and natural environmentalsurfaces such as the ground and water supplies.

[0104] A typical application for disinfection of implantable deviceswould involve soaking the device in a solution of the microbicidalcompound. Alternatively, the implantable device may be manufactured tocontain a releasable or bioactive form of the microbicidal compound,either by mechanical entrapment in the polymeric material composing thesurface of the device or by covalent chemical attachment to thepolymeric material composing the surface of the device. For treatment oftransplantable organs, the organ may be immersed in a solution of themicrobicidal agent contained in a suitable vehicle. Whole body washingcan be accomplished by thoroughly wiping the body with a solution of themicrobicidal agent, or by immersion of the body in a suitable solution.

[0105] Control of dental caries and/or gum disease may be accomplishedby washing of the oral cavity with a suitable solution of themicrobicidal agent, or by incorporation into a toothpaste used inbrushing the teeth.

[0106] Numerous medical applications and devices requiring disinfectionor decontamination are possible such as pacemakers, defibrillators,artificial hearts or parts thereof, whole body washing of infectedpatients, treatment of transplantable organs for transplantation,decontamination of surgical rooms and surgical equipment, and control ofdental caries or gum disease.

[0107] Decontamination associated with spore-forming bacteria such asBacillus anthracis, inhibitors of germination may cause damage to thespore and should be bactericidal to the vegetative cell. Thus theseinhibitors may be used to decontaminate a variety of environmentsincluding, but not limited to, environmental surfaces and drinkingwater.

[0108] In the treatment, prevention, or control of fungal and bacterialdiseases in plants and foodcrops, the inhibitor can be carried in asuitable vehicle and sprayed onto the plants to either prevent or treatfungal and/or bacterial diseases. Alternatively, application may be madeby deposition of solutions or solid preparations on the soil neargrowing plants.

[0109] Numerous medical applications requiring disinfection ordecontamination are possible. These include digestive tractdecontamination in humans related to surgery (see G. Ramsay and R. H.van Saene, “Selective gut decontamination in intensive care and surgicalpractice: where are we [Review],” World Journal of Surgery,22(2):164-70, February 1998; and G. Basha et al., “Local and systemiceffects of intraoperative whole-colon washout with 5 percentpovidone-iodine,” British Journal of Surgery. 86(2):219-26, February1999), the disinfection of, or impregnation of NADs inhibitors into,materials used in implantable devices such as intravenous catheters (seeO. Traore et al., “Comparison of in-vivo antibacterial activity of twoskin disinfection procedures for insertion of peripheral catheters:povidone iodine versus chlorhexidine,” Journal of Hospital Infection.44(2):147-50, February 2000; and T. S. Elliott, “Role of antimicrobialcentral venous catheters for the prevention of associated infections,”[Review] Journal of antimicrobial Chemotherapy. 43(4):441-6, April1999), pacemakers, defibrillators, artificial hearts or parts thereof,whole body washing of infected patients, treatment of transplantableorgans for transplantation, decontamination of surgical rooms andsurgical equipment, and control of dental caries or gum disease (see B.M. Eley, “Antibacterial agents in the control of supragingival plaque—areview,” British Dental Journal, 186(6):286-96, March 27, 1999).

[0110] It is to be understood that this invention is not limited to thespecific synthetic methods described herein. It is to be furtherunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

[0111] Ranges may be expressed herein as from “about” one particularvalue, and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment.

[0112] Throughout this application, where a chemical diagram has astraight line emanating from a chemical structure, such a linerepresents a CH₃ group. For example, in the following diagram:

[0113] o-methylbenzoic acid is represented.

[0114] The term “alkyl” as used herein refers to a branched orunbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl,decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. The term“cycloalkyl” intends a cyclic alkyl group of from three to eight,preferably five or six carbon atoms.

[0115] The term “alkoxy” as used herein intends an alkyl group boundthrough a single, terminal ether linkage; that is, an “alkoxy” group maybe defined as —OR where R is alkyl as defined above. A “lower alkoxy”group intends an alkoxy group containing from one to six, morepreferably from one to four, carbon atoms.

[0116] The term “alkylene” as used herein refers to a difunctionalsaturated branched or unbranched hydrocarbon chain containing from 1 to24 carbon atoms, and includes, for example, methylene (—CH₂—), ethylene(—CH₂—CH₂—), propylene (—CH₂—CH₂—CH₂—), 2-methylpropylene[—CH₂—CH(CH₃)—CH₂—], hexylene [—(CH₂)₆—] and the like. The term“cycloalkylene” as used herein refers to a cyclic alkylene group,typically a 5- or 6-membered ring.

[0117] The term “alkene” as used herein intends a mono-unsaturated ordi-unsaturated hydrocarbon group of 2 to 24 carbon atoms. Asymmetricstructures such as (AB)C═C(CD) are intended to include both the E and Zisomers. This may be presumed in structural formulae herein wherein anasymmetric alkene is present.

[0118] The term “alkynyl” as used herein refers to a branched orunbranched unsaturated hydrocarbon group of 1 to 24 carbon atoms whereinthe group has at least one triple bond.

[0119] The term “cyclic” as used herein intends a structure that ischaracterized by one or more closed rings. As further used herein, thecyclic compounds discussed herein may be saturated or unsaturated andmay be heterocyclic. By heterocyclic, it is meant a closed-ringstructure, preferably of 5 or 6 members, in which one or more atoms inthe ring is an element other than carbon, for example, sulfur, nitrogen,etc.

[0120] The term “bicyclic” as used herein intends a structure with twoclosed rings. As further used herein, the two rings in a bicyclicstructure can be the same or different. Either of the rings in abicyclic structure may be heterocyclic.

[0121] By the term “effective amount” of a compound as provided hereinis meant a sufficient amount of the compound to provide the desiredtreatment or preventive effect. As will be pointed out below, the exactamount required will vary from subject to subject, depending on thespecies, age, and general condition of the subject, the severity of thedisease that is being treated, the particular compound used, its mode ofadministration, and the like. Thus, it is not possible to specify anexact “effective amount.” However, an appropriate effective amount maybe determined by one of ordinary skill in the art using only routineexperimentation. It is preferred that the effective amount beessentially non-toxic to the subject, but it is contemplated that sometoxicity will be acceptable in some circumstances where higher dosagesare required.

[0122] By “pharmaceutically acceptable carrier” is meant a material thatis not biologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the compounds of the inventionwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

[0123] As used herein, “NAD synthetase enzyme” is defined as the enzymethat catalyzes the final reaction in the biosynthesis of NAD, namely,the transformation of NaAD into NAD. As used herein, the term “catalyticsites” are defined as those portions of the NAD synthetase enzyme thatbind to substrates, and cofactors, including nicotinic acid dinucleotide(NaAD), NAD, adenosine triphosphate (ATP), adenosine monophosphate(AMP), pyrophosphate, magnesium and ammonia in bacteria or microbes. Theterm “receptor site” or “receptor subsite” relates to those portions ofthe bacterial NAD synthetase enzyme in which the bacterial NADsynthetase enzyme inhibitors disclosed herein are believed to bind. Forthe purposes of this disclosure, the terms “catalytic site,” “receptorsite” and “receptor subsite” may be used interchangeably. The inhibitorsmay also inhibit the NAD synthetase enzyme by mechanisms not involvingbinding of the inhibitor to catalytic sites.

[0124] As used herein, the term “antimicrobial compound” denotes amaterial that kills or deactivates microbes so as to reduce or eliminatethe harmful effects of the bacteria on a subject or in a system.Microbes are microorganisms which are too small to be seen by the nakedeye, e.g., bacteria, fungi, viruses, and protozoa, preferably bacteriaand fungi. For example, antibacterials are known in the art as“bacteriostatic agents” or “bateriocidal agents.” The bacteria soaffected can be gram positive, gram negative or a combination thereof.The terms “antimicrobial compound” and “broad spectrum antibiotic”denote a material that kills or deactivates a wide variety of microbes,including, but not limited to, one of more of, gram positive or gramnegative bacteria, Staphylococcus aureus, Streptococcus pyogenes,Streptococcus viridans, Enterococcus, anaerobic Streptococcus,Pneumococcus, Gonococcus, Meningococcus, Mima, Bacillus anthracis, C.diphtheriae, List. monocytogenes, Streptobacillus monohiliformis,Erysipelothrix insidiosa, E. coli, A. aerogenes, A. faecalis, Proteusmirabilis, Pseudomonas aeruginosa, K pneumoniae, Salmonella, Shigella,H. influenzae, H. ducreyi, Brucella, Past. pestis, Past. tularensis,Past. multocida, V. comma, Actinobacillus mallei, Pseud. pseudomallei,Cl. tetani, Bacteroides, Fusobacterium fusiforme, M. tuberculosis,atypical mycobacteria, Actinomyces israelii, Nocardia, T. pallidum, T.pernue, Borrelia recurrentis, Peptospira, Rickettsia, and Mycoplasmapneumoniae.

[0125] In accordance with the desirability for developing improvedantimicrobials, e.g., antibacterial and antimicrobial agents, with theinvention herein novel compounds have been identified that inhibitbacterial NAD synthetase enzymatic activity. Such activity translatesinto effectiveness as bacteriocidal agents, as well as effectiveness abroad spectrum antibiotic materials. Novel compounds have been developedthat inhibit a previously unrecognized target in prokaryotic organisms,such as bacteria, to block essential biological function and therebycause bacterial death or deactivation of the microbes. Specifically, theinvention herein has identified an enzyme found in both gram positiveand gram negative bacteria, NAD synthetase enzyme, which can be utilizedas a target for drug design to provide protection from and/or treatmentfor bacterial and other microbial infections.

[0126] The NAD synthetase enzyme catalyzes the final step in thebiosynthesis of nicotinamide adenine dinucleotide (NAD). Bacterial NADsynthetase is an ammonia-dependent amidotransferase belonging to afamily of “N-type” ATP pyrophosphatases; this family also includesasparagine synthetase and argininosuccinate synthetase. NAD synthetaseenzyme catalyzes the last step in both the de novo and salvage pathwaysfor NAD⁺ biosynthesis, which involves the transfer of ammonia to thecarboxylate of nicotinic acid adenine dinucleotide (NaAD) in thepresence of ATP and Mg⁺². The overall reaction is illustrated in FIG. 1.Unlike eukaryotic NAD synthetase e.g., that found in mammals, which canutilize glutamine as a source of nitrogen, prokaryotic NAD synthetase inbacteria utilizes ammonia as the sole nitrogen source. Through x-raycrystallography and other methods, the invention has identified markeddifferences in the structures of eukaryotic and prokaryotic forms of theNAD synthetase enzyme. For example, B. subtilis NAD synthetase enzyme,which in the invention has been crystallized and used in the drug designmethodologies herein, is a dimeric material with molecular weight around60,500. In marked contrast, the eukaryotic form of NAD synthetase foundin mammals is multimeric and has a molecular weight of at least 10 timeslarger.

[0127] By utilizing the significant differences between the eukaryoticand prokaryotic forms of NAD synthetase enzyme, the invention hereinprovides novel compounds that can be utilized as antimicrobial agentsthat specifically target the prokaryotic NAD synthetase enzyme withoutsignificantly affecting a mammalian host. With the invention herein, ithas been found that by specifically inhibiting bacterial NAD synthetaseenzymatic activity, bacteria can be deprived of the energy necessary tothrive and replicate. Accordingly, through the invention disclosed andclaimed herein, antibacterial drugs may be developed that preferentiallyattack the bacteria to kill or deactivate it so as to reduce oreliminate its harmful properties, without appreciably affectingmammalian NAD synthetase enzymatic activity at the same dosage.Moreover, the invention provides methods of treating microbialinfections in a mammal, e.g., human. Because of the differences instructure between bacterial and mammalian NAD synthetase enzyme, itwould not be expected that the compounds of the invention would inhibitor otherwise affect mammalian NAD synthetase enzyme in the same manneras the compounds act on bacteria.

[0128] Without being bound by theory, through chemical analysis andx-ray crystallography methods, characterized at least two separatecatalytic subsites on the bacterial NAD synthetase enzyme in which it ispossible to bind at least one or more small molecules (“activemolecules”) have been characterized. These sites are illustrated in FIG.2.

[0129] Because of the specific structure of these catalytic sites, itmay be possible to identify small molecules that will demonstrateaffinity for at least one of the sites. Small molecules of the properconfiguration, the configuration being determined by the structure ofthe catalytic site(s), may bind with a receptor site or sites on themicrobial, e.g., bacterial NAD synthetase enzyme, thereby blocking thecatalytic activity of the enzyme. FIG. 3 illustrates a bacterial NADsynthetase enzyme in which the catalytic sites are blocked by an exampleof a compound of the present invention.

[0130] Under such circumstances, it is hypothesized that, for example,spore-forming bacteria will be unable to undergo germination andoutgrowth, and the essential cellular respiratory functions of thevegetative bacteria will be halted, thereby causing cellular death ordeactivation, e.g., gram positive and gram negative bacteria and othermicrobes will be killed or prevented from growing. Accordingly, theinvention has found that compounds that exhibit inhibitory activityagainst the bacterial NAD synthetase enzyme will also exhibittherapeutic activity as antibacterial and antimicrobial compounds, aswell as broad spectrum antibiotic materials.

[0131] With embodiments of the invention described herein, it ispossible to synthesize novel tethered dimeric compounds that exhibitactivity as microbial NAD synthetase enzyme inhibitors. By linking oneor more active molecules through a linker molecule, one or more ends ofthe tethered dimer can bind in the respective receptor sites or subsitesto thereby render the bacterial NAD synthetase enzyme inactive. Whenmore than one active molecule is used, each active molecule can be thesame or different. The term “active molecules” as used herein refers tosmall molecules that may be used alone or tethered together through alinker (tether) fragment to form a tethered dimeric compound. Further,under some circumstances, different active molecules will be more likelyto bind to different locations in the receptor site of a bacterial NADsynthetase enzyme because of the differing chemical make-up of each ofthese sites. Therefore, in one embodiment, it may be beneficial totether at least two different active molecules to each other whereineach active molecule demonstrates selective affinity for a differentsubsite in the receptor. Using the tethered dimers herein it may bepossible to drastically enhance the potency of NAD synthetase enzymeinhibition, as compared to blocking a single site on the bacterial NADsynthetase enzyme. As used herein, the term “selective affinity” meansthat the active molecule shows enhanced tendency to bind with onesubsite with the receptor in the bacterial NAD synthetase enzyme becauseof a chemical complementarity between the receptor subsite and theactive molecule. A tethered dimer compound is illustrated below.

[0132] In one embodiment, a dimeric inhibitor compound will bind with,for example, the sites of catalytic activity on the bacterial NADsynthetase enzyme, thereby preventing the production of NAD/NADH by thebacteria. By varying the length of the linker molecule, or the distancebetween the two active molecules, the affinity of the inhibitor compoundfor the NAD synthetase enzyme maybe varied.

[0133] In practice of the invention relating to the design of novel NADsynthetase enzyme inhibitor compounds, a software program can beutilized which facilitates the prediction of the binding affinities ofmolecules to proteins so as to allow identification of commerciallyavailable small molecules with the ability to bind to at least onereceptor subsite in the bacterial NAD synthetase enzyme. An example ofone such computer program is DOCK, available from the Department ofPharmaceutical Chemistry at the University of California, San Francisco.DOCK evaluates the chemical and geometric complementarity between asmall molecule and a macromolecular binding site.

[0134] The active molecules specifically disclosed herein may be used,as well as any pharmaceutically acceptable salts thereof. As noted,pharmaceutically acceptable salts of the compounds set out herein beloware also contemplated for use in this invention. Such salts are preparedby treating the free acid with an appropriate amount of apharmaceutically acceptable base. Representative pharmaceuticallyacceptable bases are ammonium hydroxide, sodium hydroxide, potassiumhydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide,ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide,ferric hydroxide, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, lysine, arginine, histidine, and the like. Thereaction is conducted in water, alone or in combination with an inert,water-miscible organic solvent, at a temperature of from about 0° C. toabout 100° C., preferably at room temperature. The molar ratio of thecompounds to base used are chosen to provide the ratio desired for anyparticular salts. For preparing, for example, the ammonium salts of thefree acid starting material—a particular preferred embodiment—thestarting material can be treated with approximately one equivalent ofpharmaceutically acceptable base to yield a neutral salt. When calciumsalts are prepared, approximately one-half a molar equivalent of base isused to yield a neutral salt, while for aluminum salts, approximatelyone-third a molar equivalent of base will be used.

[0135] Compounds prepared in accordance with the design and synthesismethods of this invention are especially attractive because they maypreferably be further optimized by incorporation of substituents oneither the active molecule and/or the linking group. These lattermodifications can also preferably be accomplished using thecombinatorial methods disclosed herein.

[0136] In a preferred embodiment, the invention provides administering abroad spectrum antibiotic to a mammal in need of such treatment orprevention. In a further preferred embodiment, the microbial infectionis a bacterial infection. In yet another embodiment of the invention,the bacterial infection is caused by a bacterium that is a gram negativeor gram positive bacteria. The bacterial infection may preferably becaused by an antibiotic resistant strain of bacteria.

[0137] Further provided by the invention herein is preferably a methodof killing a prokaryote with an amount of prokaryotic NAD synthetaseenzyme inhibitor compound to reduce or eliminate the production of NADwhereby the prokaryote is killed. A method of decreasing prokaryoticgrowth, comprising contacting the prokaryote with an amount of aprokaryotic NAD synthetase enzyme inhibitor effective to reduce oreliminate the production of NAD whereby prokaryotic growth is decreasedis also provided. In the method of killing a prokaryote, as well as inthe method of decreasing prokaryotic growth, the compound comprises oneor more compounds provided herein.

[0138] In the method of killing a prokaryote, as well as in the methodof decreasing prokaryotic growth, the prokaryote is a bacterium. Furtherpreferably, the bacterium is a gram negative or a gram positivebacteria. Still preferably, the prokaryote is an antibiotic resistantstrain of bacteria.

[0139] Also in the method of killing a prokaryote, as well as in themethod of decreasing prokaryotic growth, the NAD synthetase enzymeinhibitor is a compound that selectively binds with catalytic sites orsubsites on a bacterial NAD synthetase enzyme to reduce or eliminate theproduction of NAD by the bacteria.

[0140] In the methods discussed above, the compound is preferablyadministered by oral, rectal, intramuscular, intravenous, intravesicularor topical means of administration. The compounds of this invention canbe administered to a cell of a subject either in vivo or ex vivo. Foradministration to a cell of the subject in vivo, as well as foradministration to the subject, the compounds of this invention can beadministered orally, parenterally (e.g., intravenously), byintramuscular injection, by intraperitoneal injection, subcutaneousinjection, transdermally, extracorporeally, topically, mucosally or thelike.

[0141] Depending on the intended mode of administration, the compoundsof the present invention can be in pharmaceutical compositions in theform of solid, semi-solid or liquid dosage forms, such as, for example,tablets, suppositories, pills, capsules, powders, liquids, suspensions,lotions, creams, gels, or the like, preferably in unit dosage formsuitable for single administration of a precise dosage. The compositionswill include, as noted above, an effective amount of the selectedcomposition, possibly in combination with a pharmaceutically acceptablecarrier and, in addition, may include other medicinal agents,pharmaceutical agents, carriers, adjuvants, diluents, etc.

[0142] Parenteral administration of the compounds of the presentinvention, if used, is generally characterized by injection. Injectablescan be prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution of suspension in liquidprior to injection, or as emulsions. As used herein, “parenteraladministration” includes intradermal, subcutaneous, intramuscular,intraperitoneal, intravenous and intratracheal routes. One approach forparenteral administration involves use of a slow release or sustainedrelease system such that a constant dosage is maintained. Thesecompounds can be present in a pharmaceutically acceptable carrier, whichcan also include a suitable adjuvant. By “pharmaceutically acceptable,”it is meant a material that is not biologically or otherwiseundesirable, i.e., the material maybe administered to an individualalong with the selected compound without causing substantial deleteriousbiological effects or interacting in a deleterious manner with any ofthe other components of the composition in which it is contained.

[0143] Routes of administration for the compounds herein are preferablyin a suitable and pharmacologically acceptable formulation. Whenadministered to a human or an animal subject, the bacterial NADsynthetase enzyme inhibitor compounds of the libraries herein arepreferably presented to animals or humans orally, rectally,intramuscularly, intravenously, intravesicularly or topically (includinginhalation). The dosage preferably comprises between about 0.1 to about15 g per day and wherein the dosage is administered from about 1 toabout 4 times per day. The preferred dosage may also comprise between0.001 and 1 g per day, still preferably about 0.01, 0.05, 0.1, and 0.25,0.5, 0.75 and 1.0 g per day. Further preferably, the dosage may beadministered in an amount of about 1, 2.5, 5.0, 7.5,10.0, 12.5 and 15.0g per day. The dosage may be administered at a still preferable rate ofabout 1, 2, 3, 4 or more times per day. Further, in some circumstances,it may be preferable to administer the compound of the inventioncontinuously, as with, for example, intravenous administration. Theexact amount of the compound required will vary from subject to subject,depending on the species, age, weight and general condition of thesubject, the particular compound used, its mode of administration andthe like. Thus, it is not possible to specify an exact amount for everycompound. However, an appropriate amount can be determined by one ofordinary skill in the art using only routine experimentation given theteachings herein.

[0144] If ex vivo methods are employed, cells or tissues can be removedand maintained outside the subject's body according to standardprotocols well known in the art. The compounds of this invention can beintroduced into the cells via known mechanisms for uptake of smallmolecules into cells (e.g., phagocytosis, pulsing onto class IMHC-expressing cells, liposomes, etc.). The cells can then be infused(e.g. in a pharmaceutically acceptable carrier) or transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

[0145] It is further provided a method of disinfecting a materialcontaminated by a microbe, comprising contacting a contaminated materialwith a bacterial NAD synthetase enzyme inhibitor compound in an amountsufficient to kill or deactivate the microbe. In yet another embodiment,the compound utilized for contacting comprises one or more compoundsprovided herein.

[0146] In yet a further embodiment of the invention herein, thecompounds of the present invention are effective as disinfectantmaterials for, for example, hard or soft surfaces, fabrics, and othercontaminated materials such as those in hospitals, households, schools,nurseries, and any other location. In yet another embodiment, theinvention provides a method for disinfecting comprising contacting abacterial contaminated material with a bacterial NAD synthetase enzymeinhibitor compound.

[0147] The inhibitors of NAD synthetase according to the presentinvention can be employed in a variety of processes for the treatment ofhumans, animals and plants as well as decontamination, sterilizationand/or disinfectant techniques. The present invention further provides amethod for preventing germination of spore-forming bacteria and/or thevegetative growth of bacteria, fungi and/or molds comprisingadministering an effective amount of at least one inhibitor of NADsynthetase, e.g. prophylactically or therapeutically, e.g., to at leastone of a human, a mammal, or an animal.

[0148] The present invention further provides a method for preparing acompound of the formula A:

Ar₁—X—Ar₂—O—(CH₂)_(n)—NHCO—Q₁Ar₃  (A)

[0149] wherein Ar₁, Ar₂, and Ar₃ are independently aryl or heteroaryl,optionally substituted with one or more substituents selected from thegroup consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, halo, amino, C₁-C₆ alkylamino,C₁-C₆ dialkylamino, C₁-C₆ trialkylamino, C₁-C₆ alkylamino C₁-C₆ alkyl,C₁-C₆ dialkylamino C₁-C₆ alkyl, C₁-C₆ trialkylamino C₁-C₆ alkyl, azido,amine oxide, hydroxy, carboxyl, C₁-C₆ alkylcarbonyl, C₁-C₆ alkylcarbonylC₁-C₆ alkyl, C₁-C₆ alkylcarbonyloxy, C₁-C₆ alkylcarbonyloxy C₁-C₆ alkyl,C₁-C₆ alkyloxycarbonyl C₁-C₆ alkyl, C₁-C₆ alkyloxycarbonyl, C₁-C₆alkylthio, nitro, nitrosyl, cyano, hydroxylamino, sulfonamido, C₁-C₆dialkyl sulfonamido, C₁-C₆ alkylcarbonylamino, formyl, formylamino,mercaptyl, and heterocyclyl; optionally, a ring nitrogen atom ofheteroaryl Ar₁, Ar₂, or Ar₃ may be quaternized;

[0150] X is selected from the group consisting of a covalent bond,(CH₂)_(m)O, O(CH₂)_(m), (CH₂O)_(m), (OCH₂)_(m), (CH₂CH₂O)_(m),(OCH₂CH₂)_(m), C(═O)O, OC(═O), OC(═O)O, (CH₂)_(m)S, S(CH₂)_(m),(CH₂S)_(m), (SCH₂)_(m), NH, NR, +NR₂, C(═O)NH, C(═O)NR, NHC(═O),NRC(═O), CH(OH), and CH(OR), wherein R is C₁-C₆ alkyl and m is 0-5;

[0151] Q₁ is (i) a C₁-C₆ alkylenyl, C₁-C₆ alkylenyl carbonyloxy C₁-C₆alkyl, or C₁-C₆ alkylenyl carbonylamino C₁-C₆ alkyl group, optionallyhaving a substituent selected from the group consisting of amino, C₁-C₆alkylamino, C₁-C₆ haloalkylamino, C₁-C₆ haloalkyl C₁-C₆ alkyl amino,C₁-C₆ hydroxyalkylamino, C₁-C₆ hydroxyalkyl C₁-C₆ alkylamino, C₁-C₆dialkylamino, C₁-C₆ trialkylamino, and a heterocyclic containing anitrogen atom which may be optionally quaternized;

[0152] and n is from 1 to 15;

[0153] comprising (i) providing a compound of the formula B:

Ar₁—X—Ar₂—O—(CH₂)_(n)—NH₂  (B)

[0154] and (ii) reacting the compound of formula B with a compound offormula C:

HOOC—Q₁Ar₃  (C);

[0155] wherein Q₁ is optionally protected.

[0156] In an embodiment, the compound of formula B may be prepared byreacting a compound of formula D: Ar₁—X—Ar₂—OH (D) with a compound offormula E: Hal—(CH₂)_(n)—NPhth (E); wherein “Hal” stands for a halogenatom and “NPhth” stands for phthalidimide linked to (CH₂)_(n) at thenitrogen atom, to obtain a compound of formula F:

Ar₁—X—Ar₂—O—(CH₂)_(n)—NPhth  (F);

[0157] and hydrolyzing the compound of formula F.

[0158] In accordance with another embodiment, the present inventionprovides a method for preparing a compound of the formula G:

Ar₁—X—Ar₂—O—(CH₂)_(n)—O—Q₁Ar₃  (G)

[0159] wherein Ar₁, Ar₂, and Ar₃ are independently aryl or heteroaryl,optionally substituted with one or more substituents selected from thegroup consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, halo, amino, C₁-C₆ alkylamino,C₁-C₆ dialkylamino, C₁-C₆ trialkylamino, C₁-C₆ alkylamino C₁-C₆ alkyl,C₁-C₆ dialkylamino C₁-C₆ alkyl, C₁-C₆ trialkylamino C₁-C₆ alkyl, azido,amine oxide, hydroxy, carboxyl, C₁-C₆ alkylcarbonyl, C₁-C₆ alkylcarbonylC₁-C₆ alkyl, C₁-C₆ alkylcarbonyloxy, C₁-C₆ alkylcarbonyloxy C₁-C₆ alkyl,CL—C₆ alkyloxycarbonyl C₁-C₆ alkyl, C₁-C₆ alkyloxycarbonyl, C₁-C₆alkylthio, nitro, nitrosyl, cyano, hydroxylamino, sulfonamido, C₁-C₆dialkyl sulfonamido, C₁-C₆ alkylcarbonylamino, formyl, formylamino,mercaptyl, and heterocyclyl; optionally, a ring nitrogen atom ofheteroaryl Ar₁, Ar₂, or Ar₃ may be quaternized;

[0160] X is selected from the group consisting of a covalent bond,(CH₂)_(m)O, O(CH₂)_(m), (CH₂O)_(m), (OCH₂)_(m), (CH₂CH₂O)_(m),(OCH₂CH₂)_(m), C(═O)O, OC(═O), OC(═O)O, (CH₂)_(m)S, S(CH₂)_(m),(CH₂S)_(m), (SCH₂)_(m), NH, NR, +NR₂, C(═O)NH, C(═O)NR, NHC(═O),NRC(═O), CH(OH), and CH(OR), wherein R is C₁-C₆ alkyl and m is 0-5;

[0161] Q₁ is (i) a C₁-C₆ alkylenyl, C₁-C₆ alkylenyl carbonyloxy C₁-C₆alkyl, or C₁-C₆ alkylenyl carbonylamino C₁-C₆ alkyl group, optionallyhaving a substituent selected from the group consisting of amino, C₁-C₆alkylamino, C₁-C₆ haloalkylamino, C₁-C₆ haloalkyl C₁-C₆ alkyl amino,C₁-C₆ hydroxyalkylamino, C₁-C₆ hydroxyalkyl C₁-C₆ alkylamino, C₁-C₆dialkylamino, C₁-C₆ trialkylamino, and a heterocyclic containing anitrogen atom which may be optionally quaternized;

[0162] and n is from 1 to 15;

[0163] comprising (i) providing a compound of the formula H:

Ar₁—X—Ar₂—O—(CH₂)_(n)—OH  (H)

[0164] and (ii) reacting the compound of formula H with a compound offormula J:

[0165]   HO—Q₁Ar₃  (J);

[0166] wherein Q₁ is optionally protected.

[0167] In accordance with an embodiment, the compound of formula H maybe prepared by reacting a compound of formula D:

Ar₁—X—Ar₂—OH  (D)

[0168] with a compound of formula K:

Hal—(CH₂)_(n)—OH  (K)

[0169] wherein “Hal” stands for a halogen atom, e.g., cl, Br, or I toobtain a compound of formula L:

Ar₁—X—Ar₂—O—(CH₂)_(n)—OH  (L).

[0170] In a preferred embodiment, the present invention provides amethod for preparing the above compounds wherein n is from 7 to 13relating to compounds of formulas A and G. In accordance with anembodiment, Ar₁, Ar₂, and Ar₃ are aryl, particularly phenyl. In anembodiment, X is CH₂O. In accordance with an embodiment of the method,Q₁ is a C₁-C₆ alkylenyl, optionally having a substituent selected fromthe group consisting of amino, C₁-C₆ alkylamino, C₁-C₆ haloalkylamino,C₁-C₆ haloalkyl C₁-C₆ alkyl amino, C₁-C₆ hydroxyalkylamino, C₁-C₆hydroxyalkyl C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆ trialkylamino,and a heterocyclic containing a nitrogen atom which may be optionallyquaternized, preferably Q₁ is C₁-C₃ alkylenyl, having a substituentselected from the group consisting of amino, C₁-C₆ alkylamino. C₁-C₆dialkylamino, and C₁-C₆ trialkylamino.

[0171] The present invention further provides a pharmaceuticalcomposition comprising at least one of the compounds described alongwith a pharmaceutically acceptable carrier.

[0172] The pharmaceutically acceptable carriers described herein, forexample, vehicles, adjuvants, excipients, or diluents, are well-known tothose who are skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the active compound and one which hasno detrimental side effects or toxicity under the conditions of use.

[0173] The choice of carrier will be determined in part by theparticular active agent, as well as by the particular method used toadminister the composition. Accordingly, there is a wide variety ofsuitable formulations of the pharmaceutical composition of the presentinvention. The following formulations for oral, aerosol, parenteral,subcutaneous, intravenous, intraarterial, intramuscular,interperitoneal, intrathecal, rectal, and vaginal administration aremerely exemplary and are in no way limiting.

[0174] Formulations suitable for oral administration can consist of (a)liquid solutions, such as an effective amount of the compound dissolvedin diluents, such as water, saline, or orange juice; (b) capsules,sachets, tablets, lozenges, and troches, each containing a predeterminedamount of the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations may include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, and the polyethylene alcohols andpolyethylene glycols, either with or without the addition of apharmaceutically acceptable surfactant, suspending agent, or emulsifyingagent. Capsule forms can be of the ordinary hard- or soft-shelledgelatin type containing, for example, surfactants, lubricants, and inertfillers, such as lactose, sucrose, calcium phosphate, and cornstarch.Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such carriers as are known in the art.

[0175] The compounds of the present invention, alone or in combinationwith other suitable components, can be made into aerosol formulations tobe administered via inhalation. These such formulations. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5 to about15% by weight. Suitable surfactants include polyethylene sorbitan fattyacid esters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

[0176] The compounds of the present invention may be made intoinjectable formulations. The requirements for effective pharmaceuticalcarriers for injectable compositions are well known to those of ordinaryskill in the art. See Pharmaceutics and Pharmacy Practice, J. B.Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed.,pages 622-630 (1986).

[0177] The present invention further provides a method for treating orpreventing a microbial (e.g., bacterial or fungal) infection in a mammalcomprising administering to said mammal an effective amount of at leastone of the compounds described above. The present invention alsoprovides a method for treating or preventing tuberculosis.

[0178] The present invention further provides a method for combatingagroterrorism involving an infective agent on an object comprisingtreating the object with an amount of a compound effective to inhibitthe NAD synthetase of the infective agent. Agroterrorism is defined asthe intentional introduction of animal or plant pests or the cultivationor production of pathogenic bacteria, fungi, parasites, protozoans,viruses, or their toxic products for the purpose of causing poultry,livestock, crop, soil, or human disease, poisoning, or death. This couldoccur through introducing pests intended to kill food crops, spreadingvirulent disease among confined feedlots where animals are given highprotein rations for preparing them for slaughter, poisoning civil oragricultural water sources or food supplies, or using food-bornepathogens to cause human disease. Food-borne pathogens aremicroorganisms that cause illness through the ingestion of food.

[0179] The following examples further illustrate the invention but, ofcourse, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0180] This Example illustrates a method of preparing compounds of thepresent invention in accordance with an embodiment of the invention.

[0181] Experimental Procedures

[0182] Melting points were determined using an Electrothermal 9100apparatus and are uncorrected. IR spectra were taken with BruckerVector-22 and Bomen MB-104 instruments. All ¹H and ¹³C NMR spectra wererecorded on a Brucker 300 MHz spectrometer using TMS as internalstandard. The values of chemical shifts (δ) are given in ppm andcoupling constants (J) in Hz. Elemental analyses were performed byAtlantic Microlab, Norcross, Ga. Reactions were monitored by TLC(Whatmann, Silica gel, UV254, 25 μM plates) and flash columnchromatography was done using ‘BAKER’ silica gel (40 μM) in solventsystems indicated. The solvents used for reactions were purchased asanhydrous in Sure-Seal™ bottles from Aldrich chemical company. All otherreagents were used as received.

[0183] Synthesis of Compound 1364 (Scheme 1)

[0184] Compound 2

[0185] To a solution of 4-(benzyloxy)phenol 1 (0.40 g, 2.0 mmol) in 10mL of DMF was added solid NaH (60% in mineral oil, 88 mg, 2.2 mmol), andthe mixture was stirred at r.t for 30 min under a nitrogen atmosphere.N-(8-Bromooctyl) phthalimide (0.74 g, 2.2 mmol) was added and themixture stirred at room temperature for 3 h. The reaction mixture wasquenched with water (20 mL) and extracted with EtOAc (2×20 mL). Theorganic layer was washed with water (2×10 mL) and brine (10 mL), Removalof solvent from the dried (Na₂SO₄) extract gave the crude product. Itwas crystallized from MeOH to afford 2 (0.71 g, 78% yield) as a whitesolid. m.p: 74-75° C. (MeOH)., ¹H-NMR (CDCl₃) δ 1.26-1.48 (m, 8H),1.59-1.79 (m, 4H), 3.67 (t, 2H, J=7.28 Hz), 3.88 (t, 2H, J=6.53 Hz),5.00 (s, 2H), 6.81 (d, 2H, J=9.14 Hz), 6.89 (d, 2H, J=9.15 Hz),7.27-7.45 (m, 5H), 7.66-7.73 (m, 2H) and 7.81-7.86 (m, 2H); ¹³CNMR(CDCl₃) δ 25.9, 26.7, 28.5, 29.1, 29.2, 29.3, 37.9, 68.4, 70.6, 115.3,115.7, 123.1, 127.4, 127.8, 128.5, 132.1, 133.8, 137.3, 152.7, 153.4 and168.4; IR (neat): 1693 cm⁻¹; MS (ES⁺): 458 (M+1).

[0186] Compound 3

[0187] To a solution of 2 (11.1 g, 24.3 mmol) in CH₂Cl₂ (120 mL) andMeOH (16 mL) was added anhydrous hydrazine (2.29 mL, 72.9 mmol) at r.t.under a nitrogen atmosphere. The reaction mixture was stirred overnightat r.t. Formation of white precipitate, which is a by-product occurred.The precipitate was filtered and washed with NH₄OH saturated CHCl₃:MeOH(10:1). The filtrate was evaporated to get rid of methanol, thenre-dissolved in NH₄OH saturated CHCl₃ (400 mL), washed with 1N NaOH(3×60 mL), water (2×60 mL), and brine (2×60 mL). After drying overNa₂SO₄, the organic layer was concentrated to about 250 mL, and 1N HCl(60 mL) was added to the above solution, resulting in the formation of awhite precipitate. This was filtered and washed with water and CHCl₃.After drying under vacuum hydrochloride salt 3 (6.8 g, 77% yield) wasobtained as a white solid., m.p. 180-182° C., ¹H-NMR (DMSO-d₆) δ1.22-1.44 (m, 8H), 1.48-1.61 (m, 2H), 1.61-1.72 (m, 2H), 2.67-2.81 (m,2H), 3.86 (t, 2H, J=6.38 Hz), 5.02 (s, 2H), 6.83 (d, 2H, J=9.10 Hz),6.92 (d, 2H, J=9.09 Hz), 7.28-7.45 (m, 5H) and 7.98 (bs 3H); ¹³CNMR(CDCl₃) δ 25.5, 25.8, 26.9(2C), 28.5, 28.6, 28.8, 67.7, 69.6, 115.2,115.6, 127.6, 127.7, 128.4, 137.4, 152.2 and 152.8; IR (neat): 3440cm⁻¹; MS (ES⁺): 328 (M+).

[0188] Compound 4

[0189] Compound 3 (hydrochloride salt) (0.95 g, 2.6 mmol) was suspendedin CH₂Cl₂ (15 mL) and cooled to 0° C. Et₃N (0.44 mL, 3.12 mmol) wasadded and stirred for 5 min. Then N,N-dimethyl-L-phenylalanine (0.61 g,3.12 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.60g, 3.12 mmol) and DMAP (0.036 g, 0.3 mmol) were added. The reactionmixture was stirred at r.t. overnight. The reaction mixture was dilutedwith CH₂Cl₂ (50 mL) and washed with 1M NaHCO₃ (2×20 mL), water (2×20mL), and brine (20 mL). Removal of solvent from the dried (Na₂SO₄)extract gave the crude product, which was purified by flash columnchromatography (20×4 cm) over silica gel using 1% MeOH in CHCl₃ toafford the pure amide 4 (1.35 g, 103% yield) as a white solid., mp:61-62° C., ¹H-NMR (CDCl₃) δ 1.18-1.35 (m, 6H), 1.35-1.49 (m, 4H),1.67-1.81 (m, 2H), 2.29 (s, 6H), 2.81-2.92 (m, 1H), 3.08-3.26 (m, 4H),3.88 (t, 2H, J=6.52 Hz), 4.99 (s, 2H), 6.73 (bs, 1H), 6.81 (d, 2H,J=9.17 Hz), 6.89 (d, 2H, J=9.18 Hz) and 7.12-7.45 (m, 10H); ¹³CNMR(CDCl₃) δ 25.8, 26.7, 29.1, 29.2, 29.3, 29.5, 32.7, 38.9, 42.2, 68.4,70.5, 71.0, 115.2, 115.6, 125.9, 127.3, 127.7, 128.2, 128.4, 129.1,137.2, 140.0, 152.7, 153.4 and 171.9; MS (ES+): 503 (M+1); Anal. Calcdfor C₃₂H₄₂N₂O₃.0.5H₂O: C, 75.16; H, 8.47; N 5.48, found: C, 75.15; H,8.23 and N 5.42.

[0190] Compound 1364

[0191] To a solution of compound 4 (0.096 g, 0.19 mmol) in anhydrous DME(3 mL) was added iodomethane (0.35 mL, 5.6 mmol). The reaction mixturewas heated at 80° C. for 12 h with stirring and cooled to roomtemperature. After evaporation, the crude product was purified by flashsilica gel column (10×2 cm) chromatography over silica gel using,stepwise, CHCl₃ MeOH (30:1 followed by 10:1) to afford pure 1364 (0.095g, 77% yield).m.p.: 98-99° C., ¹H-NMR (CDCl₃) δ 0.95-1.31 (m, 8H),1.31-1.45 (m, 2H), 1.66-1.79 (m, 3H), 2.85-2.99 (m, 1H), 3.03-3.15 (m,1H), 3.15-3.33 (m, 2H), 3.48 (s, 9H), 3.88 (t, 3H, J=6.52 Hz), 5.00 (s,2H), 5.74 (dd, 1H, J₁=11.2 Hz, J₂=4.57 Hz), 6.83 (d, 2H, J=9.17 Hz),6.90 (d, 2H, J=9.17 Hz), 7.22-7.43 (m, 10H) and 7.78 (t, 1H, J=5.58 Hz);¹³CNMR (CDCl₃) δ 20; IR (neat):3245, 1679 cm⁻¹; MS (ES⁺): 517(M+); Anal.Calcd for C₃₃H₄₅₁N₂O₃: C, 61.49; H, 7.04; N 4.35; found: C, 61.20; H,6.89 and N, 4.23.

[0192] Synthesis of 1439 (Scheme 2)

[0193] Compound 5.

[0194] To a solution of 4-(benzyloxy)phenol 1 (2.4 g, 12 mmol) in DMF(32 mL) was added sodium hydride (0.528 g, 13.2 mmol, 60% in mineraloil), and the mixture was stirred under N₂ at r.t. for 30 min.8-Bromo-1-octanol (2.25 mL, 13.2 mmol) was added and the reactionmixture was further stirred at r.t. for 5 h. TLC (30% EtOAc in hexanes)showed that reaction was complete. After being quenched with saturatedammonium chloride solution and ice, the mixture was extracted with EtOAc(3×60 mL). The combined organic layer was then washed with IN. NaOHsolution (2×40 mL), water (2×40 mL) and brine (2×40 mL). After drying(Na₂SO₄) the organic layer was evaporated and concentrated to around 20mL, when a white solid began precipitating. The mixture was cooled,filtered, and the filter washed with hexane to give 2.2 g white solid.The filtrate was further cooled to give another 0.8 g of 5 (76.9% yield)as a white solid.,mp. 94-95° C. ¹H-NMR (CDCl₃) δ 1.28-1.51 (m, 8H),1.51-1.63 (m, 3H), 1.69-1.81 (m, 2H), 3.62 (t, 2H, J=6.58 Hz), 3.88 (t,2H, J=6.52 Hz), 5.00 (s, 2H), 6.82 (d, 2H, J=9.09 Hz), 6.89 (d, 2H,J=9.21 Hz) and 7.26-7.45 (m, 5H); ¹³C-NMR (CDCl₃) δ 25.6, 25.9, 29.3,32.6, 62.9, 68.5, 70.6, 115.3, 115.7, 127.4, 127.8, 128.4, 137.2, 152.7and 153.4; IR (KBr): 3303 cm⁻¹; MS (ES⁺): 329 (M+1); Anal. Calcd forC₂₁H₂₈O₃: C, 76.78; H, 8.60; found: C, 76.64 and H, 8.58.

[0195] Compound 6

[0196] To a cooled solution (0° C.) of alcohol 5 (1 g, .3.05 mmol) inCH₂Cl₂ (40 mL) was added 2,6-lutidine (0.46 mL, 3.955 mmol), followed bytriflic anhydride (0.62 mL, 3.687 mmol). After stirred at 0° C. for 15min. TLC (EtOAc:Hexanes 1:3) showed the reaction is complete. Thereaction mixture was then washed with water (2×20 mL), brine (20 mL) anddried (Na₂SO₄). The solvent was completely removed and the producttriflate was dried at high vacuum for 10 min. Triflate was thendissolved in CH₂Cl₂ (10 mL) and added in 10 minutes to a solution ofN-Boc phenyl alaminol (1.53 g, 6.095 mmol) and NaH (0.305 g, 60% inmineral oil, 7.625 mmol) in CH₂Cl₂ (30 mL) kept at 0° C. Reactionbubbled vigorously. It was stirred for 5 minutes and 18-crown-6 (0.081g, 0.307 mmol) was added and the reaction mixture was allowed to attainroom temperature and stirred at room temp for 30 minutes. TLC (25% EtOAcin hexanes) showed that the reaction is complete. Reaction was thenwashed with water (2×20 mL) and brine (20 mL). Removal of solvent fromthe dried (Na₂SO₄) extract gave the crude product which was purified byf column chromatography over silica gel (20×4 cm) using 10% EtOAc inhexanes as eluent to afford the pure ether 6 (1.39 g, 81.28% yield) as awhite solid. mp.65-66° C. ¹H-NMR (CDCl₃) δ 1.28-1.39 (m, 6H), 1.42 (s,9H), 1.39-1.51 (m, 2H), 1.51-1.64 (m, 2H), 1.69-1.81 (m, 2H), 2.75-2.94(m, 2H), 3.23-3.32 (m, 2H), 3.32-3.45 (m, 2H), 3.88 (t, 2H, J=6.52 Hz),4.88 (d, 1H, J=8.04 Hz), 4.98 (s, 2H), 6.81 (d, 2H J=9.26 Hz), 6.88 (d,2H, J=9.21 Hz) and 7.15-7.43 (m, 10H); ¹³CNMR (CDCl₃) δ 25.9, 26.1, 28.3(2C), 29.29, 29.33, 29.5, 37.7, 51.5, 68.4, 70.3, 70.5, 71.1, 79.1,115.2, 115.6, 126.1, 127.3, 127.7, 128.2, 128.4, 129.4, 137.2, 138.2,152.7, 153.4 and 155.3; IR (neat):1685, 3373 cm⁻¹; MS (ES⁺): 562 (M+1);Anal. Calcd for C₃₅H₄₇NO₅: C, 74.83; H, 8.43; N 2.49; found: C, 74.59;H, 8.39 and N, 2.56.

[0197] Compound 7

[0198] To a solution of Boc-protected ether 6 (1.00 g, 1.782 mmol) inCH₂Cl₂ (5 mL) at room temperature a solution of TFA (5 mL) in CH₂Cl₂ (5mL) was added and stirred at room temperature for 30 min. TLC (10% MeOHin CHCl₃) showed that the reaction is complete. Solvent and TFA wereremoved completely under vacuum and residue was dissolved in CH₂Cl₂ (20mL). It was washed with sat. Na₂CO₃ (2×10 mL), water (2×10 mL) and brine(10 mL). Removal of solvent from the dried (Na₂SO₄) extract gave thecrude product. Purified by column chromatography over silica gel (15×3cm) using 10% MeOH in CHCl₃ to obtain the pure amine 7 (0.71 μg, 86.51%yield) as a colorless oil. ¹H-NMR (CDCl₃) δ 1.28-1.38 (m, 6H), 1.38-1.50(m, 4H), 1.50-1.64 (m, 2H), 1.67-1.79 (m, 2H), 2.52 (dd, 1H, J₁=13.19Hz, J₂=7.29 Hz), 2.76 (dd, 1H, J₃=13.34 Hz, J₂=4.45 Hz), 3.15-3.27 (m,2H), 3.33-3.48 (m, 3H), 3.86 (t, 2H, J=6.44 Hz), 4.96 (s, 2H), 6.80 (d,2H, J=9.12 Hz), 6.87 (d, 2H, J=8.92 Hz) and 7.15-7.43 (m, 10H); ¹³CNMR(CDCl₃) δ 25.8, 25.9, 29.2 (2C), 29.3, 29.5, 40.6, 52.2, 68.3, 70.4,71.1, 75.2, 115.1, 115.6, 126.1, 127.3, 127.7, 128.3, 128.4, 129.1,137.2, 138.8, 152.6 and 153.3; MS (ES⁺): 462 (M+1); Anal. Calcd forC₃₀H₃₉NO₃: C, 78.05; H, 8.52; N 3.03; found: C, 77.83; H, 8.56 and N,3.02.

[0199] Compound 1439

[0200] To a solution of the amine 7 (0.7 g, 1.518 mm01) in DME (15 mL)was added potassium carbonate (1.25 g, 9.057 mmol) and iodomethane (1.4mL, 22.4 mmol). The reaction mixture was stirred at room temperatureovernight. TLC (10% MeOH in CHCl₃) showed that the reaction is complete.Precipitation of the product was observed. CHCl₃ was added to thereaction mixture until all the product went into a solution. K₂CO₃ wasremoved by filtration through celite 521. The filtrate was concentratedon a rotary evaporator until solid began precipitating out. This wasfiltered and washed with DME and ethyl acetate to obtain pure 1439 (0.518 g, 54.08% yield) as a white solid., mp. 128-129° C. ¹H-NMR (CDCl₃) δ1.29-1.40 (m, 6H), 1.40-1.52 9m, 2H), 1.52-1.63 (m, 2H), 1.69-1.81 (m,2H), 3.12 (t, 1H, J=12.26 Hz), 3.22-3.43 (m, 4H), 3.58 (s, 9H),3.84-3.95 (m, 3H), 4.25-4.33 (m, 1H), 5.00 (s, 2H), 6.81 (d, 2H, J=9.15Hz), 6.90 (d, 2H, J=9.14 Hz) and 7.21-7.45 (m, 10H); ¹³CNMR (CDCl₃) δ25.9, 26.1, 29.18, 29.2, 29.24, 29.3, 31.3, 53.4, 65.1, 68.4, 70.5,71.7, 73.8, 115.2, 115.6, 127.3, 127.5, 127.7, 128.4, 129.0, 129.4,134.7, 137.1, 152.7 and 153.3; MS (ES⁺): 504 (M+); Anal. Calcd forC₃₃H₄₆INO₃: C, 62.75; H, 7.34; N 2.22; found: C, 62.40; H, 7.17 and N,2.17.

[0201] Synthesis of Guanidine 1503

[0202] Compound 8

[0203] Compound 3 (free amine, 1.0 g, 3.05 mmol), N-Boc-L-phenylalanine(0.89 g, 3.35 mmol), 1-[3-[(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (0.72 g, 3.66 mmol), and DMAP (0.036 g, 0.30 mmol) wasdissolved in 20 mL of anhydrous CH₂Cl₂. The mixture was stirred at roomtemperature for 3 h, diluted with CHCl₃ (50 mL) and washed with 5%NaHCO₃ solution (2×30 mL) followed by brine (1×30 mL). The organic layerwas dried over sodium sulfate and evaporated under reduced pressure. Theproduct was purified by flash silica gel column chromatography usinghexanes/CHCl₃/MeOH (3/1/0.1) to afford 8 (1.5 g, 86% yield) as a whitesolid. m.p.: 108-110° C.; ¹H NMR (CDCl₃) δ 1.17-1.45 (m, 10H), 1.41 (s,9H), 1.69-1.82 (m, 2H), 2.96-3.18 (m, 4H), 3.89 (t, 2H, J=6.5 Hz), 4.26(q, 1H, J=7.6 Hz,), 5.01 (s, 2H), 5.09 (bs, 1H, —NH), 5.67 (bs, 1H,—NH), 6.82 (d, 2H, J=9.2 Hz), 6.90 (d, 2H, J=9.2 Hz), 7.19-7.41 (m,10H); ¹³C NMR (CDCl₃). δ 26.19, 26.87, 28.48, 29.35, 29.44,29.53, 29.54,38.98, 39.62, 68.69, 70.87, 115.55, 115.98, 127.67, 128.07, 128.74,128.86, 129.52, 137.50, 153.03, 153.66, 171.11.; MS (ES+) 575 (M+1), 475(M+1-Boc); (ES−) 573 (M−1).

[0204] Compound 9

[0205] To a solution of compound 8 (1.5 g. 2.61 mmol) in 10 mL ofanhydrous CH₂Cl₂ was added 5 mL of trifluoroacetic acid in 5 mL ofanhydrous CH₂Cl₂ at room temperature under argon atmosphere. The mixturewas stirred for 30 min and the reaction was quenched by an addition of10 g of solid NaHCO₃. The mixture was partitioned between water (50 mL)and CHCl₃ (2×100 mL). The organic layer was dried over sodium sulfateand evaporated under reduced pressure. The product was crystallized fromether to afford 9 (1.2 g, 97% yield) as a white solid. m.p.: 82-84° C.;¹H NMR (CDCl₃) δ 1.30-1.57 (m, 10H), 1.67-1.77 (m, 2H), 2.68 (dd, 1H,J=9.1, 13.6 Hz), 3.15-3.27 (m, 3H), 3.54 (dd, 1H, J=4.3, 9.1 Hz), 3.85(t, 2H, J=6.5 Hz), 4.95 (s, 2H), 6.79 (d, 2H, J=9.2 Hz), 6.87 (d, 2H,J=9.2 Hz), 7.17-7.37 (m, 10H); ¹³C NMR (CDCl₃) δ 25.93, 26.77, 29.15,29.21, 29.28, 38.98, 41.01, 56.37, 68.40, 70.50, 115.26, 115.68, 126.66,127.37, 127.77, 128.44, 128.57, 129.25, 137.25, 137.95, 152.74, 153.39,174.00; MS (ES+) 475 (M+1).

[0206] Compound 10

[0207] To a stirred solution of compound 9 (0.1 g, 0.21 mmol) in DMF wasadded triethylamine (0.073 mL, 0.52 mmol) and HgCl₂ (0.095 g, 0.35mmol). The mixture was cooled down to 0° C. andbis-Boc-S-methyl-isothiourea (0.091, 0.31 mmol) was added at once. Themixture was stirred for 3 h and after the filtration of resulting whitesolid, the filtrate was partitioned between 5% NaHCO₃ (50 mL) and EtOAc(3×50 mL). The organic layer was dried over sodium sulfate andevaporated under reduced pressure. The product was purified by silicagel column chromatography using hexanes/EtOAc (15/1 to 7/1) to afford 10(0.14 g, 93% yield) as a colorless oil; ¹H NMR (CDCl₃) δ 1.20-1.55 (m,10H), 1.48 (s, 18H), 1.69-1.77 (m, 2H), 3.05-3.18 (m, 4H), 3.88 (t, 2H,J=6.5 Hz), 4.67 (q, 1H, J=7.2 Hz), 5.00 (s, 2H), 6.42 (1H, pseudo t,—NH), 6.82 (d, 2H, J=9.3 Hz), 6.90 (d, 2H, J=9.3 Hz), 7.18-7.44 (m,10H), 8.81 (d, 1H, J=7.2 Hz, —NH), 11.31 (s, 1H, —NH); ¹³C NMR (CDCl₃) δ26.14, 26.28, 26.84, 28.13, 28.39, 29.30, 29.38, 29.48, 37.76, 39.55,56.06, 68.60, 70.78, 79.45, 83.60, 115.47, 115.90, 126.99, 127.61,128.00, 128.63, 128.67, 129.66, 137.02,137.44,152.79,152.95,153.59,155.90, 163.14, 170.21; MS (ES+) 717 (M+1); (ES−) 715(M−1).

[0208] Compound 1503

[0209] To a solution of compound 10 (0.3 g. 0.42 mmol) in 2 mL ofanhydrous CH₂Cl₂ was added 1 mL of trifluoroacetic acid in 1 mL ofanhydrous CH₂Cl₂ at room temperature under argon atmosphere. The mixturewas stirred for 1 h and evaporated under reduced pressure. The residuewas partitioned between 5% Na₂CO₃ (50 mL) and EtOAc (3×50 mL). Theorganic layer was dried over sodium sulfate and evaporated under reducedpressure. The product was purified by silica gel column chromatographyusing CHCl₃/MeOH/30% NH₄OH (10/1/0.1) to afford 0.17 g of free base of1503 as a amorphous solid (79% yield). A solution of free base of 1503(50 mg) in 3 mL of anhydrous CH₂Cl₂ was added 50 μL of trifluoroaceticacid and the mixture was evaporated with anhydrous ether (5×10 mL). Theresulting white crystal was suspended with ether and collected byfiltration to give 1503 (0.045 g, 74% yield). m.p.: 108-113° C.; ¹H NMR(free base, CDCl₃) δ 1.11-1.55 (m, 10H), 1.68-1.77 (m, 2H), 2.85-3.23(m, 4H), 3.86 (t, 2H, J=6.5 Hz), 4.82 (bs, 1H), 4.97 (s, 2H), 6.80 (d,2H, J=9.2 Hz), 6.88 (d, 2H, J=9.2 Hz), 7.21-7.41 (m, 10H), 7.95 (bs, 2H,—NH₂), 8.23 (bs, 1H, —NH); ¹³C NMR (CDCl₃) δ 26.17, 26.81, 28.73, 29.31,29.38, 29.53, 40.13, 68.66, 70.80, 115.49, 115.94, 127.65, 128.02,128.69, 128.96, 129.30, 135.04, 137.45, 152.98, 153.62, 157.09, 171.22;MS (ES+) 517 (M+1).

[0210] Synthesis of Cyclic Guanidine Compound 1686 (Scheme 4)

[0211] Compound 1686

[0212] A solution of compound 9 (0.05 g, 0.105 mmol) and2-methylthio-2-imidazoline hydroiodide (0.14 g 0.580 mmol) in anhydrousCH₃CN was refluxed for 2 days. The mixture was cooled down andevaporated under reduced pressure. The residue was

[0213] suspended with CH₂Cl₂ and any solid was removed by filtration.The filtrate was concentrated and purified by silica gel columnchromatography using [NH₄OH saturated CHCl₃/MeOH (100/1 to 10/1) andcrystallization from ether to give 30 mg of 1686 (0.03 g, 53% yield) asa white solid. ¹H NMR (free base, CDCl₃, 8 ppm) 1.18-1.52 (m, 10H),1.65-1.79 (m, 2H), 2.74 (dd, 1H, J=9.2, 13.1 Hz), 3.10-3.41 (m, 7H),3.52 (bs, 1H, —NH), 3.89 (t, 2H, J=6.5 Hz), 3.85-3.94 (m, 1H), 4.82 (bs,1H), 5.01 (s, 2H), 6.78 (d, 2H, J=9.2 Hz), 6.90 (d, 2H, J=9.2 Hz), 7.22(m, 1H, —NH), 7.26-7.41 (m, 10H); ¹³C NMR (CDCl₃, 6 ppm) 26.19, 27.03,29.41, 29.47, 29.53, 29.59, 39.29, 40.85, 43.69, 62.06, 68.71, 70.85,115.53, 115.96, 126.69, 127.68, 128.05, 128.69, 128.73, 129.64, 137.49,139.37, 153.00, 153.64, 160.14, 173.33.; MS (ES+) 543 (M+1); (ES−) 541(M−1).

[0214] Synthesis of Guanidine Compound 1679 (Scheme 5)

[0215] Compound 11

[0216] To a solution of 7 (0.30 g, 0.65 mmol) in anhydrous DMF (4 mL),Et₃N (0.35 mL, S 2.63 mmol) and 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (0.208 g, 0.717 mmol)were added and stirred for 5 min at r.t. HgCl₂ (0.194 g, 0.715 mmol) wasadded to the reaction mixture and stirring continued at r.t. for 30 min.TLC (10% MeOH in CHCl₃) showed that the reaction is complete. Dilutedwith EtOAC (20 mL) and the white solid formed was filtered off throughcelite 521. Filtrate was washed with water (3×5 mL), brine (5 mL) anddried (Na₂SO₄). Removal of solvent gave the crude product which waspurified by column (15×2 cm) chromatography over silica gel using 10%EtOAc in hexanes as eluent to afford the pure product 11 (0.218 g, 92.8%yield); ¹H-NMR (CDCl₃) δ 1.27-1.43 (m, 8H), 1.47 (s, 9H), 1.50 (m, 9H),1.53-1.65 (m, 2H), 1.67-1.81 (m, 2H), 2.83-3.02 (m, 2H), 3.25-3.35 (m,2H), 3.39 (t, 2H, J=6.30 Hz), 3.88 (t, 2H, J=6.53 Hz), 4.41-4.54 (M,1H), 4.98 (s, 2H), 6.81 (d, 2H, J=9.18 Hz), 6.88 (d, 2H, J=9.21 Hz),7.15-7.43 (m, 10H), 8.66 (d, 1H, J=8.34 Hz) and 11.48 (s, 1H); ¹³CNMR(CDCl₃) δ 25.9, 26.0, 27.9, 28.2, 29.2, 29.3, 29.4, 29.5, 37.2, 51.4,68.3, 69.4, 70.4, 71.1, 78.7, 82.6, 115.2, 115.6, 126.2, 127.3, 127.7,128.1, 128.4, 129.5, 137.2, 137.9, 152.6, 152.8, 153.3, 155.5 and 163.6;MS (ES⁺): 704 (M+1); Anal. Calcd for C₄₁H₅₇N₃O₇: C, 69.96; H, 8.16; N5.97; found: C, 69.91; H, 8.10 and N, 5.93.

[0217] Compound 1679

[0218] To a solution of 11 (0,1 6 g, 0.227 mmol) in CH₂Cl₂ (2 mL) asolution of TFA (2 mL) in CH₂Cl₂ (2 mL) was added and stirred at r.t.for 3.5 h. TLC (in 10% MeOH in CHCl₃) showed that the reaction iscomplete. Solvent and TFA were completely removed, redissolved in CH₂Cl₂(20 mL), washed with sat. Na₂CO₃ (3×5 mL), water (2×5 mL) and brine (5mL). Removal of solvent from the dried (Na₂SO₄) extract gave the crudeproduct which was purified by column (10×2 cm) chromatography oversilica gel using 10% MeOH in CHCl₃ as eluent to afford the pureguanidine derivative 1679 (0.082 g, 71.65% yield); 1H-NMR (CDCl₃) δ1.17-1.45 (m, 8H), 1.45-1.61 (m, 2H), 1.65-1.81 (m, 2H), 2.73-2.87 (m,1H), 2.87-3.04 (m, 1H), 3.25-3.43 (m, 3H), 3.43-3.56 (m, 1H), 3.65-3.80(m, 1H), 3.86 (t, 2H, J=6.46 Hz), 4.96 (s, 2H), 6.80 (d, 2H, J=9.18 Hz),6.88 (d, 2H, J=8.97 Hz), 7.03 (bs, 1H), 7.16-7.49 (m, 12H, Ar—H and NH₂)and 8.09 (d, 1H, 6.54 Hz); ¹³CNMR (CDCl₃) δ 25.6, 25.7,29.0 (2C),29.1(2C), 37.1, 55.1, 68.2,70.4,71.6, 73.9, 115.1, 115.5, 126.8, 127.2,127.6, 128.3, 128.5, 128.8, 136.4, 137.1, 152.5, 153.2 and 158.8; IR(neat):3155, 3259, 3329 cm⁻¹; MS (ES⁺): 504 (M+1); Anal. Calcd forC₃₃H₄₂F₃N₃O₅ (TFA salt): C, 64.17; H, 6.85; N 6.80; found: C, 64.68; H,7.06 and N, 6.78.

EXAMPLE 2

[0219] This example illustrates some of the properties of compounds ofthe present invention.

[0220] Antimicrobial Testing (P. aeruginosa, E. coli, B. subtilis, S.aureus, S. aureus (MCR), A. niger and M. flavescens.)

[0221] The MIC test procedures conformed to the present protocol fromthe National Committee for Clinical Laboratory Standards (NCCLS), andwere designed to provide basic antimicrobial data on compounds based onthe MIC of the active component.

[0222] The test compounds were solubilized, diluted, and pipetted induplicate into 10 mL sterile culture tubes and dried under vacuum.

[0223] Challenge organisms, specified were grown overnight at 37° C. inthe appropriate medium (i.e., Mueller-Hinton Broth). These pure brothcultures were diluted 1:1,000 and 2.0 mL were added to the test compoundtubes.

[0224] Appropriate media controls, challenge organism viabilitycontrols, and antibiotic control dilutions (i.e., ampicillin andnystatin), were prepared in the same manner as the test compounds andrun against the challenge organisms.

[0225] The cultures were incubated overnight at 37° C. and MIC's inμg/ml were indicated by visual determination of the first clear tube.

[0226] The minimum inhibitory concentration (MIC) was defined as theconcentration of test compound that completely inhibited growth of thechallenge organism.

[0227] Antimicrobial Testing (B. anthracis).

[0228] To determine the MIC of each compound in liquid bacterial culturemedium against spores of Bacillus anthracis Sterne 34F2, an MIC range of128 μg/mL −0.0156 μg/mL was tested in triplicate using a 48-well, tissueculture plate. Dilutions (of up to 1:7.8) of each compound (stockconcentration 1.0 mg/mL in 100% methanol) were made in 2× M-H broth.Spores were suspended to 1×10⁶ spores/400 μl in filter-sterilized MilliQwater. Potency of Various Compounds to Inhibit Gram-Negative BacterialGrowth or the Growth of Two Fungi In Vitro Compound No. P aeruginosa E.coli C. albicans A. niger 1197 >50 >50 <1.56    <6.25 1364 >50; >50<50; >50  <1.56; <12.56 >50; >50 1420 >50 >50 <3.13 >50 1423 >50 >50<3.13 <50 1439 >50   <12.5 <3.13 <25 1447 >50; >50 >50: <50 <6.25;<3.13 >50; >50 1450 >50 >50 <6.25 >50 1503 >50 >50 >50    >50Ciprofloxacin   <5.0   <5.0 — — Doxycycline <30    <1.56 — —Amphotericin — — <1.56    <1.56 B

[0229] Potency of Various Compounds to Inhibit Bacterial Growth In VitroCompound S. aureus B. M. No. B. subtilis S. aureus (MCR) anthracisflavescens 1197 <0.78 <0.78 <0.78 — — 1364  <0.39;  <0.78;  <0.78;— >50    <6.25 <2.5  <1.56 1391 <3.13 — <1.56 — — 1420 <0.39 <1.56 <0.78— — 1423 <0.20 <1.56 <1.56 — — 1439 <0.39 <0.78 <1.56 4; 4  <12.5  1447<1.56  <1.56;  <1.56; — <6.25 <0.78 <0.78 <1.56 1450 <3.13 <3.13 <3.13 —<3.13 1484 <3.13 <0.78 — 1503  <0.78; <0.78  <0.78; 8; 16 >50    <3.13<1.56 1505 <0.78 — <0.78 — — 1594 >50    — <6.25 8; 32 — 1617 <12.5  —<6.25 16; — — 1685 <3.13 — <0.78 4; 4  — Cipro- <5.0; <0.5 <5.0  <5.0; 0.25;  floxacin <0.5  0.125 Doxycycline <1.56 <1.56 <30    — Ampicillin<0.5; <0.2 >50    —

[0230] Values are reported as μg/mL and represent the Minimal InhibitoryConcentration (MIC) for each assay.

[0231] Where multiple values are shown (B.subtilis, S. aureus, S. aureus(MCR)), these represent two or more tests performed.

[0232] For assays using B. anthracis, the approximate MICs weredetermined for various compounds in a standard broth dilution assayusing bacterial growth media or by visually inspecting the samples forturbidity (appearance of bacterial outgrowth) when bacteria werecultured in mammalian cell culture media.

[0233] Cytotoxicity in Murine 3T3 Cells.

[0234] Methods

[0235] Prior to conducting the assay, cell number, serum concentration,and medium conditions were optimized. Then using the assay conditions asdescribed below, cells were seeded on day 1 and allowed to adhere for atleast 1 hour. Test articles were=added to achieve a final concentrationof 10, 50 and 100 μM in the cultures (Note: in an initial assay, aconcentration of 500 μM was included but due to solubility issues aswell as marked cytotoxicity, this concentration was excluded from thefinal assay). The initial solubilization of the test articles was in50:50 methanol:water (v/v). (Note: Compound 1364 went into solution onday 1, but precipitated on day 2. The organic solvent was increased from50% to 66%.(v/v)). Compound 1439 never went into solution at 50%organic. Compound 1503b went into solution at 50% organic. Maximumconcentration of methanol did not exceed 1.6% in the final assay. On day1, compounds 1439 and 1364 required sonication before solubility wasreached). The cells plus compound were incubated overnight at 37° C.,under a 5% CO₂/95% O₂ atmosphere. On day 2, the cells in the positivecontrol wells were lysed with 0.9% Triton X-100 for 45 minutes toestablish maximum levels of LDH release. The plates were centrifuged at250× g for 5 minutes, the supernatants transferred to a new assay plate,and the LDH was measured. The assay plates were read at OD 490 nm.Experimental Conditions Assay Conditions Culture Medium DMEM, 10% calfserum Control: Maximum LDH release Lysis with Triton X-100 at 0.9%Control: Spontaneous LDH Cells with no compound added release Control:LDH standard Bovine heart LDH (included in kit) Incubation Conditions37° C. for 20 hrs; 5% CO₂/95% O₂ Compound Concentrations 10 μM, 50 μM,100 μM Enzyme Assay Promega Cytotox 96 Cytotoxicity Assay

[0236] Cytotoxicity in Rabbit Primary Renal Cells.

[0237] Isolation of Proximal Tubules and Culture Conditions.

[0238] Rabbit renal proximal tubules were isolated using the iron oxideperfusion method and grown in 35-mm tissue culture dishes under improvedconditions. The cell culture medium was a 1:1 mixture of DMEM/Ham's F-12(without D-glucose, phenol red, or sodium pyruvate) supplemented with 15mM HEPES buffer, 2.5 mM L-gluatmine, 1 μM pyridoxine HCL, 15 mM sodiumbicarbonate, and 6 mM lactate. Hydrocortisone (50 nM), selenium (5ng/mL), human transferrin (5 μg/mL), bovine insulin (10 nM) andL-ascorbic acid-2-phosphate (50 μM) were added to fresh culture mediumimmediately prior to daily media change.

[0239] Treatment of RPRC.

[0240] All numbered compounds were diluted in methanol and the finalconcentration of methanol in RPRC was less then 0.1% (v/v). 4-BOP alsowas dissolved in methanol while TMAI was dissolved in ethanol andciprofloxacin was dissolved in media. These concentrations of solventsdid not cause any increases in RPRC death alone.

[0241] Measurement of RPRC Death.

[0242] Cell death was monitored in RPRC by assessment of both annexin Vand PI staining using flow cytometry. Briefly, RPRC were exposed to theindicated concentrations of compounds for 24 hr. Media was removed andRPRC washed twice with phosphate-buffered saline (PBS) and incubated ina binding buffer (10 mM HEPES, 140 mM NaCL, 5 mM KCL, 1 MM MgCl₂, 1.8 mMCaCl₂, pH=7.4) containing annexin V-FITC (1 μmol) and PI (25 μg/mL).After a 10 min incubation, RPRC were washed three times in the bindingbuffer and were released from the monolayers by gentle scrapping with arubber policeman. Annexin V and PI staining were measured using aBectonDickson FacsCalibur flow cytometer (San Jose, Calif.). An equalnumber of cells (10,000) were counted for sample and apoptotic cellswere defined as those that stained positive for annexin V-FITC only.RPRC undergoing necrotic cell death stained for PI only. Late apoptoticcells (RPRC dying initially by apoptosis and/or necrotic cell death thatexhibited more extensive degradation of the plasma membrane over time)were defined as those that stained positive for both annexin V and PI.

[0243] Data Analysis.

[0244] RPRC isolated from one rabbit represented one experiment (n=1).Data were analyzed and are reported as means ±standard error of the meanof at least 3 separate experiments. Effect of Various Compounds onMammalian Cell Growth In Vitro % CELLULAR % CELLULAR % Compound NECROSISNECROSIS APOPTOSIS No. (FIBROBLASTS)¹ (RPRC)² (RPRC)³ Control  2 ± 1  7± 1 TMAI —  2 ± 1  5 ± 1 Cipro —  4 ± 1  9 ± 3 1617 — 18 ± 2 23 ± 3 1594— 29 ± 4 13 ± 2 1439 73 62 ± 2 10 ± 1 1364 62 63 ± 2 15 ± 3

EXAMPLE 3

[0245] This Example illustrates the NAD synthetase enzyme inhibitingactivity of some compounds of the present invention.

[0246] The coupled assay—production of NAD was monitored throughconversion to NADH by alcohol dehydrogenase. [NADH] was monitored by 2parallel methods: the change in absorbance at 340 nm, and fluorescenceat 460 nm (excitation 320 nm). The assay condition were as follows:Total volume=200 μL; 58.5 mM HEPPS, pH 8.5; 18.5 mM NH₄Cl; 9.75 mMMgCl₂; 1% (v/v) EtOH; 0.3% BOG (w/v); 40 μg/n ADH; 0.1 NaAD; 0.2 mM ATP;2.0 μg/mL NAD synthetase; 2.5% (v/v) DMSO. Controls were included fordetermining inhibitor background, precipitation, and ADH inhibition.IC₅₀ (μM) Compound No. Mean SD SEM N 1126 36.2 1 1127 23.0 1.8 0.37 23 1168 36.2 1 1169 36.2 1 1182 42.1 3.2 1.84 3 1186 46.1 2 1197 20.8 2.70.57 23  1264 38.1 2 1290 36.2 1 1291 36.2 1 1292 36.2 1 1294 36.2 11321 22.0 1.8 1.27 2 1322 26.5 4.8 3.41 2 1323 48.2 1 1324 50.7 1 133619.4 1.1 0.57 4 1337 20.3 2.0 0.99 4 1338 23.0 2.6 1.28 4 1339 19.8 1.70.85 4 1340 19.3 1.6 0.79 4 1358 46.6 0.00 2 1359 42.2 6.6 3.80 3 136448.4 14.7  4.90 9 1369 29.2 3.0 1.71 3 1370 16.9 0.9 0.45 4 1371 19.11.2 0.67 3 1387 20.5 5.8 2.58 5 1388 14.5 4.0 2.32 3 1389 45.5 8.5 4.933 1390 14.2 3.1 0.85 13  1391 11.5 2.9 0.60 24  1393 36.7 4.9 2.81 31394 22.5 4.0 2.32 3 1396 15.1 2.8 1.25 5 1397 16.9 3.0 1.76 3 1398 16.02.2 1.29 3 1401 >100 μM 1405 18.8 2 1408 30.5 5.3 3.03 3 1420 31.3 9.65.54 3 1421 34.8 2.8 1.98 2 1422 27.9 3.9 2.73 2 1423 32.9 12.8  7.36 31431 18.2 1.9 1.09 3 1432 16.6 3.3 0.99 11  1439 22.7 5.1 1.81 8 144221.4 1.9 1.38 2 1443 27.9 17.0  9.80 3 1447 22.1 6.2 2.06 9 1448 30.70.5 0.32 2 1450 31.7 9.0 4.03 5 1451 34.2 0.6 0.43 2 1454 23.9 2.7 1.902 1456 17.0 2.3 0.67 12  1475 22.6 1 1477 14.0 2.9 0.93 10  1478 15.810.5  3.17 11  1479 18.6 0.6 0.42 2 1482 26.2 8.3 5.88 2 1483 16.0 1.70.85 4 1484 14.8 6.3 1.89 11  1485 34.8 1 1486 37.8 1 1491 19.9 5.6 1.6811  1494 12.5 5.6 1.68 11  1495 33.4 19.1  7.21 7 1498 39.4 1 1499 26.81 1501 38.2 1 1502 35.6 1 1503 12.7 3.4 0.72 23  1505  6.6 1.5 0.41 14 1593 18.1 3.3 2.33 2 1594 25.0 6.8 4.77 2 1596 28.0 1 1597 21.3 1 159944.0 1 1600 28.7 8.6 2.73 10  1603 34.9 12.2  3.39 13  1604 21.1 4.12.92 2 1605 21.0 1 1606 39.1 1 1608 26.3 1 1609 20.7 1 1610 30.4 1 161123.3 8.0 5.68 2 1612 26.2 12.0  8.48 2 1613 19.1 3.0 2.12 2 1614 23.1 11615 22.3 7.1 5.05 2 1616 38.5 1 1617 31.4 14.8  8.55 3 1619 22.3 2 162021.9 2 1621 25.5 14.8  14.81  1 1622 34.2 1 1623 33.9 1 1624 31.2 1 162921.1 1 1632 18.5 1 1633 20.4 1 1634 21.2 1 1635 35.1 1 1636 19.0 2.51.75 2 1637 23.3 1 1644 23.9 1 1645 18.1 5.4 3.80 2 1650  >50 μM 165147.7 1652 17.9 1653 19.5 1658 16.8 2.4 1.72 2 1661 23.7 7.8 3.20 6 166223.2 5.5 1.95 8 1663 18.4 1 1664 19.9 1 1665 19.6 2.5 0.79 10  1666 27.71 1678 36.5 1 1679 10.4 2.2 0.75 9 1680 10.1 1.2 0.37 11  1681 12.9 1.40.46 9 1682 17.6 3.7 1.38 7 1683 20.2 1 1685  9.9 1.7 0.60 8

[0247] All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

[0248] The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

[0249] Preferred embodiments of this invention are described herein,including the best mode known to the inventors for carrying out theinvention. Variations of those preferred embodiments may become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

What is claimed is:
 1. A compound of formula (I): Ar₁—X—Ar₂—Y—L—Z—Q  (I)wherein Q is Q₁Ar₃ or Ar₃Q₁; Ar₁, Ar₂, and Ar₃ are independently aryl orheteroaryl, optionally substituted with one or more substituentsselected from the group consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆haloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, halo, amino,C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆ trialkylamino, C₁-C₆alkylamino C₁-C₆ alkyl, C₁-C₆ dialkylamino C₁-C₆ alkyl, C₁-C₆trialkylamino C₁-C₆ alkyl, azido, amine oxide, hydroxy, carboxyl, C₁-C₆alkylcarbonyl, C₁-C₆ alkylcarbonyl C₁-C₆ alkyl, C₁-C₆ alkylcarbonyloxy,C₁-C₆ alkylcarbonyloxy C₁-C₆ alkyl, C₁-C₆ alkyloxycarbonyl C₁-C₆ alkyl,C₁-C₆ alkyloxycarbonyl, C₁-C₆ alkylthio, nitro, nitrosyl, cyano,hydroxylamino, sulfonamido, C₁-C₆ dialkyl sulfonamido, C₁-C₆alkylcarbonylamino, formyl, formylamino, mercaptyl, and heterocyclyl;optionally, a ring nitrogen atom of heteroaryl Ar₁, Ar₂, or Ar₃ may bequaternized; X, Y, and Z are independently selected from the groupconsisting of a covalent bond, (CH₂)_(m)O, O(CH₂)_(m), (CH₂O)_(m),(OCH₂)_(m), (CH₂CH₂O)_(m), (OCH₂CH₂)_(m), C(═O)O, OC(═O), OC(═O)O,(CH₂)_(m)S, S(CH₂)_(m), (CH₂S)_(m), (SCH₂)_(m), NH, NR, ^(+NR) ₂,C(═O)NH, C(═O)NR, NHC(═O), NRC(═O), CH(OH), and CH(OR), wherein R isC₁-C₆ alkyl and m is 0-5; L is {(CR₁R₂)_(q)—(W)_(t)—(CR₃R₄)_(r)}_(p),wherein R₁-R₄ are independently selected from the group consisting of H,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxy C₁-C₆ alkyl, halo, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino,azido, hydroxy, aldehyde, C₁-C₆ acetal, C₁-C₆ ketal, C₁-C₆alkylcarbonyl, C₁-C₆ alkylcarbonyl C₁-C₆ alkyl, C₁-C₆ alkylcarbonyloxy,C₁-C₆ alkylcarbonyloxy C₁-C₆ alkyl, C₁-C₆ alkylthio, nitro, nitrosyl,cyano, sulfonamido, C₁-C₆ alkylcarbonylamino, and heterocyclyl; W is amoiety selected from the group consisting of alicyclic ring, aromaticring, heterocyclic ring, combinations of alicyclic, heterocyclic, and/oraromatic rings, C₂-C₆ alkenyl, dienyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy,C₂-C₆ alkenyloxy, C₂-C₆ alkynyloxy, anhydrido, enol, ketene, amino,imino, hydrazinyl, epoxy, episulfide, amido, amine oxide, urea,urethane, ester, thioester, carbonate, carbonyl, thiocarbonyl, sulfonyl,diazo, sulfonamido, ether oxygen, ether sulfur, thionyl, silyl,peroxide, lactam, lactone, phenylene, monosaccharide, dri-, tri-, andhigher polysaccharides, nucleic acid, amino acid, phosphonyl,phosphoryl, and combinations thereof; q, r, and t are independently0-20; q, r, and t are not simultaneously 0; and p is 1-6; L, optionally,further including O, N, or S; and Q₁ is (i) a C₁-C₆ alkylenyl, C₁-C₆alkylenyl carbonyloxy C₁-C₆ alkyl, or C₁-C₆ alkylenyl carbonylaminoC₁-C₆ alkyl group, optionally having one or more substituents selectedfrom the group consisting of amino, C₁-C₆ alkylamino, C₁-C₆haloalkylamino, C₁-C₆ haloalkyl C₁-C₆ alkyl amino, C₁-C₆hydroxyalkylamino, C₁-C₆ hydroxyalkyl C₁-C₆ alkylamino, C₁-C₆dialkylamino, C₁-C₆ trialkylamino, and heterocyclic containing anitrogen atom which may be optionally quaternized, (ii) a C₂-C₆alkylenyl; (iii) methylenyl with the proviso that Z is other thancovalent bond or O(C═O) when Q is Q₁Ar₃ wherein Ar₃ is a phenyl parasubstituted with amino, methylamino, dimethylamino, or trimethylamino orAr₃ is a pyridyl or N-methylpyridyl; (iv) a covalent bond with theproviso that when Ar₃ is pyridyl, N-methylpyridyl, or phenyl parasubstituted with trimethylaminomethyl group, Z is other than a covalentbond or O(C═O); (v) a group containing amidine or guanidine functionwherein the amidine or guanidine may be optionally N-substituted with aC₁-C₆ alkyl; or (vi) a zwitterion; or a pharmaceutically acceptable saltthereof.
 2. The compound of claim 1, wherein Ar₁, Ar₂, and Ar₃ areindependently aryl including 1-3 aromatic rings.
 3. The compound ofclaim 2, wherein Ar₁, Ar₂, and Ar₃ are independently phenyl orsubstituted phenyl.
 4. The compound of claim 1, wherein Ar₁, Ar₂, andAr₃ are independently heteroaryl including 1-3 rings one or more ofwhich include O, N, or S.
 5. The compound of claim 3, wherein Ar₁ isphenyl or phenyl substituted with one or more substituents selected fromthe group consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl,C₁-C₆ hydroxyalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, halo, amino, C₁-C₆alkylamino, C₁-C₆ dialkylamino, C₁-C₆ trialkylamino, C₁-C₆ alkylaminoC₁-C₆ alkyl, C₁-C₆ dialkylamino C₁-C₆ alkyl, C₁-C₆ trialkylamino C₁-C₆alkyl, azido, amine oxide, hydroxy, carboxyl, C₁-C₆ alkylcarbonyl, C₁-C₆alkylcarbonyl C₁-C₆ alkyl, C₁-C₆ alkylcarbonyloxy, C₁-C₆alkylcarbonyloxy C₁-C₆ alkyl, C₁-C₆ alkyloxycarbonyl C₁-C₆ alkyl, C₁-C₆alkyloxycarbonyl, C₁-C₆ alkylthio, nitro, nitrosyl, cyano,hydroxylamino, sulfonamido, C₁-C₆ dialkyl sulfonamido, C₁-C₆alkylcarbonylamino, formyl, formylamino, mercaptyl, and heterocyclyl. 6.The compound of claim 5, wherein Ar₁ is phenyl or phenyl substitutedwith one or more substituents selected from the group consisting ofC₁-C₆ alkoxy, halo, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, azido,C₁-C₆ alkylcarbonyloxy, C₁-C₆ alkylthio, nitro, cyano, sulfonamido,C₁-C₆ dialkyl sulfonamido, C₁-C₆ alkylcarbonylamino, and heterocyclyl.7. The compound of claim 3, wherein Ar₂ is phenyl, optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, halo, amino, C₁-C₆ alkylamino,C]-C₆ dialkylamino, C₁-C₆ trialkylamino, C₁-C₆ alkylamino C₁-C₆ alkyl,C₁-C₆ dialkylamino C₁-C₆ alkyl, C₁-C₆ trialkylamino C₁-C₆ alkyl, azido,amine oxide, hydroxy, carboxyl, C₁-C₆ alkylcarbonyl, C₁-C₆ alkylcarbonylC₁-C₆ alkyl, C₁-C₆ alkylcarbonyloxy, C₁-C₆ alkylcarbonyloxy C₁-C₆ alkyl,C₁-C₆ alkyloxycarbonyl C₁-C₆ alkyl, C₁-C₆ alkyloxycarbonyl, C₁-C₆alkylthio, nitro, nitrosyl, cyano, hydroxylamino, sulfonamido, C₁-C₆dialkyl sulfonamido, C₁-C₆ alkylcarbonylamino, formyl, formylamino,mercaptyl, and heterocyclyl.
 8. The compound of claim 3, wherein Ar₂ isindolyl or indolyl substituted with one or more substituents selectedfrom the group consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl,C₁-C₆ hydroxyalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, halo, amino, C₁-C₆alkylamino, C₁-C₆ dialkylamino, C₁-C₆ trialkylamino, C₁-C₆ alkylaminoC₁-C₆ alkyl, C₁-C₆ dialkylamino C₁-C₆ alkyl, C₁-C₆ trialkylamino C₁-C₆alkyl, azido, amine oxide, hydroxy, carboxyl, C₁-C₆ alkylcarbonyl, C₁-C₆alkylcarbonyl C₁-C₆ alkyl, C₁-C₆ alkylcarbonyloxy, C₁-C₆alkylcarbonyloxy C₁-C₆ alkyl, C₁-C₆ alkyloxycarbonyl C₁-C₆ alkyl, C₁-C₆alkyloxycarbonyl, C₁-C₆ alkylthio, nitro, nitrosyl, cyano,hydroxylamino, sulfonamido, C₁-C₆ dialkyl sulfonamido, C₁-C₆alkylcarbonylamino, formyl, formylamino, mercaptyl, and heterocyclyl. 9.The compound of claim 1, wherein Ar₃ is phenyl, indolyl, or pyridyl,optionally substituted with one or more substituents selected from thegroup consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, halo, amino, C₁-C₆ alkylamino,C₁-C₆ dialkylamino, C₁-C₆ trialkylamino, C₁-C₆ alkylamino C₁-C₆ alkyl,C₁-C₆ dialkylamino C₁-C₆ alkyl, C₁-C₆ trialkylamino C₁-C₆ alkyl, azido,amine oxide, hydroxy, carboxyl, C₁-C₆ alkylcarbonyl, C₁-C₆ alkylcarbonylC₁-C₆ alkyl, C₁-C₆ alkylcarbonyloxy, C₁-C₆ alkylcarbonyloxy C₁-C₆ alkyl,C₁-C₆ alkyloxycarbonyl C₁-C₆ alkyl, C₁-C₆ alkyloxycarbonyl, C₁-C₆alkylthio, nitro, nitrosyl, cyano, hydroxylamino, sulfonamido, C₁-C₆dialkyl sulfonamido, C₁-C₆ alkylcarbonylamino, formyl, formylamino,mercaptyl, and heterocyclyl.
 10. The compound of claim 9, wherein Ar₃ isphenyl, optionally substituted with one or more substituents selectedfrom the group consisting of C₁-C₆ alkoxy and C₁-C₆ trialkylamino. 11.The compound of claim 9, wherein Ar₃ is indolyl.
 12. The compound ofclaim 11, wherein Q is Ar₃Q₁.
 13. The compound of claim 12, wherein Q₁is C₁-C₆ alkylenyl carbonyloxy C₁-C₆ alkyl, optionally having a C₁-C₆trialkylamino
 14. The compound of claim 12, wherein Q₁ is trimethylaminoethylenyl carbonyloxy t-butyl.
 15. The compound of claim 12, wherein Q₁is C₁-C₆ alkylenyl, optionally having a C₁-C₆ trialkylamino or aheterocyclic containing a quaternized nitrogen atom.
 16. The compound ofclaim 12, wherein Q₁ is a covalent bond.
 17. The compound of claim 12,wherein Q₁ is a zwitterion.
 18. The compound of claim 12, wherein Q₁ isa group containing amidine or guanidine function wherein the amidine orguanidine may be optionally N-substituted with a C₁-C₆ alkyl.
 19. Thecompound of claim 1, wherein t is
 0. 20. The compound of claim 19,wherein R₁-R₄ are H.
 21. The compound of claim 20, wherein q and r areindependently 1-7.
 22. The compound of claim 1, wherein said compound isselected from the group consisting of:

wherein X⁻ or I⁻ is a pharmaceutically acceptable anion
 23. The compoundof claim 22, wherein the pharmaceutically acceptable anion is iodide.24. A compound of the formula A—B—(CH₂)_(n)—O—CO—CH₂—Ph (NMe₃)⁺I⁻,wherein A is a phenyl or indole, optionally substituted with a benzyloxygroup; B is a covalent bond or oxygen atom; n is 1-15; and r is apharmaceutically acceptable anion.
 25. The compound of claim 24,selected from the group consisting of

wherein I⁻ is a pharmaceutically acceptable anion.
 26. The compound ofclaim 25, wherein the a pharmaceutically acceptable anion is iodide. 27.A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 28. A method for treating orpreventing a microbial infection in a mammal comprising administering tosaid mammal an effective amount of a compound of claim
 1. 29. Apharmaceutical composition comprising a compound of claim 24 and apharmaceutically acceptable carrier.
 30. A method for treating orpreventing a microbial infection in a mammal comprising administering tosaid mammal an effective amount of a compound of claim
 24. 31. A methodfor treating or preventing a microbial infection in a mammal comprisingadministering to said mammal an effective amount of a compound thatinhibits the enzymatic activity of the microbial NAD synthetase.
 32. Amethod for preparing a compound of the formula A:Ar₁—X—Ar₂—O—(CH₂)n—NHCO—Q₁Ar₃  (A) wherein Ar₁, Ar₂, and Ar₃ areindependently aryl or heteroaryl, optionally substituted with one ormore substituents selected from the group consisting of C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆ alkoxy C₁-C₆alkyl, halo, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆trialkylamino, C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆ dialkylamino C₁-C₆alkyl, C₁-C₆ trialkylamino C₁-C₆ alkyl, azido, amine oxide, hydroxy,carboxyl, C₁-C₆ alkylcarbonyl, C₁-C₆ alkylcarbonyl C₁-C₆ alkyl, C₁-C₆alkylcarbonyloxy, C₁-C₆ alkylcarbonyloxy C₁-C₆ alkyl, C₁-C₆alkyloxycarbonyl C₁-C₆ alkyl, C₁-C₆ alkyloxycarbonyl, C₁-C₆ alkylthio,nitro, nitrosyl, cyano, hydroxylamino, sulfonamido, C₁-C₆ dialkylsulfonamido, C₁-C₆ alkylcarbonylamino, formyl, formylamino, mercaptyl,and heterocyclyl; optionally, a ring nitrogen atom of heteroaryl Ar₁,Ar₂, or Ar₃ may be quaternized; X is selected from the group consistingof a covalent bond, (CH₂)_(m)O, O(CH₂)_(m), (CH₂O)_(m), (OCH₂)_(m),(CH₂CH₂O)_(m), (OCH₂CH₂)_(m), C(═O)O, OC(═O), OC(═O)O, (CH₂)_(m)S,S(CH₂)_(m), (CH₂S)_(m), (SCH₂)_(m), NH, NR, ⁺NR₂, C(═O)NH, C(═O)NR,NHC(═O), NRC(═O), CH(OH), and CH(OR), wherein R is C₁-C₆ alkyl and m is0-5; Q₁ is (i) a C₁-C₆ alkylenyl, C₁-C₆ alkylenyl carbonyloxy C₁-C₆alkyl, or C₁-C₆ alkylenyl carbonylamino C₁-C₆ alkyl group, optionallyhaving a substituent selected from the group consisting of amino, C₁-C₆alkylamino, C₁-C₆ haloalkylamino, C₁-C₆ haloalkyl C₁-C₆ alkyl amino,C₁-C₆ hydroxyalkylamino, C₁-C₆ hydroxyalkyl C₁-C₆ alkylamino, C₁-C₆dialkylamino, C₁-C₆ trialkylamino, and a heterocyclic containing anitrogen atom which may be optionally quaternized; and n is from 1 to15; comprising (i) providing a compound of the formula B:Ar₁—X—Ar₂—O—(CH₂)_(n)—NH₂  (B) and (ii) reacting the compound of formulaB with a compound of formula C: HOOC—Q₁Ar₃  (C); wherein Q₁ isoptionally protected.
 33. The method of claim 32, wherein the compoundof formula B is prepared by reacting a compound of formula D:Ar₁—X—Ar₂—OH  (D) with a compound of formula E: Hal—(CH₂)_(n)—NPhth  (E)wherein “Hal” stands for a halogen atom and “NPhth” stands forphthalidimide linked to (CH₂)_(n) at the nitrogen atom, to obtain acompound of formula F: Ar₁—X—Ar₂—O—(CH₂)_(n)—NPhth  (F); and hydrolyzingthe compound of formula F.
 34. The method of claim 32, wherein n is from7 to
 13. 35. The method of claim 33, wherein n is from 7 to
 13. 36. Themethod of claim 32, wherein Ar₁, Ar₂, and Ar₃ are phenyl.
 37. The methodof claim 32, wherein X is CH₂O.
 38. The method of claim 32, wherein Q₁is a C₁-C₆ alkylenyl, optionally having a substituent selected from thegroup consisting of amino, C₁-C₆ alkylamino, C₁-C₆ haloalkylamino, C₁-C₆haloalkyl C₁-C₆ alkyl amino, C₁-C₆ hydroxyalkylamino, C₁-C₆ hydroxyalkylC₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆ trialkylamino, and aheterocyclic containing a nitrogen atom which may be optionallyquaternized.
 39. A method for preparing a compound of the formula G:Ar₁—X—Ar₂—O—(CH₂)_(n)—O—Q₁Ar₃  (G) wherein Ar₁, Ar₂, and Ar₃ areindependently aryl or heteroaryl, optionally substituted with one ormore substituents selected from the group consisting of C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆ alkoxy C₁-C₆alkyl, halo, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆trialkylamino, C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆ dialkylamino C₁-C₆alkyl, C₁-C₆ trialkylamino C₁-C₆ alkyl, azido, amine oxide, hydroxy,carboxyl, C₁-C₆ alkylcarbonyl, C₁-C₆ alkylcarbonyl C₁-C₆ alkyl, C₁-C₆alkylcarbonyloxy, C₁-C₆ alkylcarbonyloxy C₁-C₆ alkyl, C₁-C₆alkyloxycarbonyl C₁-C₆ alkyl, C₁-C₆ alkyloxycarbonyl, C₁-C₆ alkylthio,nitro, nitrosyl, cyano, hydroxylamino, sulfonamido, C₁-C₆ dialkylsulfonamido, C₁-C₆ alkylcarbonylamino, formyl, formylamino, mercaptyl,and heterocyclyl; optionally, a ring nitrogen atom of heteroaryl Ar₁,Ar₂, or Ar₃ may be quaternized; X is selected from the group consistingof a covalent bond, (CH₂)_(m)O, O(CH₂)_(m), (CH₂O)_(m), (OCH₂)_(m),(CH₂CH₂O)_(m), (OCH₂CH₂)_(m), C(═O)O, OC(═O), OC(═O)O, (CH₂)_(m)S,S(CH₂)_(m), (CH₂S)_(m), (SCH₂)_(m), NH, NR, ⁺NR₂, C(═O)NH, C(═O)NR,NHC(═O), NRC(═O), CH(OH), and CH(OR), wherein R is C₁-C₆ alkyl and m is0-5; Q₁ is (i) a C₁-C₆ alkylenyl, C₁-C₆ alkylenyl carbonyloxy C₁-C₆alkyl, or C₁-C₆ alkylenyl carbonylamino C₁-C₆ alkyl group, optionallyhaving a substituent selected from the group consisting of amino, C₁-C₆alkylamino, C₁-C₆ haloalkylamino, C₁-C₆ haloalkyl C₁-C₆ alkyl amino,C₁-C₆ hydroxyalkylamino, C₁-C₆ hydroxyalkyl C₁-C₆ alkylamino, C₁-C₆dialkylamino, C₁-C₆ trialkylamino, and a heterocyclic containing anitrogen atom which may be optionally quaternized; and n is from 1 to15; comprising (i) providing a compound of the formula H:Ar₁—X—Ar₂—O—(CH₂)_(n)—OH  (H) and (ii) reacting the compound of formulaH with a compound of formula J: HO—Q₁Ar₃  (J); wherein Q₁ is optionallyprotected.
 40. The method of claim 39, wherein the compound of formula His prepared by reacting a compound of formula D: Ar₁—X—Ar₂—OH  (D) witha compound of formula K: Hal—(CH₂)_(n)—OH  (K) wherein “Hal” stands fora halogen atom, to obtain a compound of formula L:Ar₁—X—Ar₂—O—(CH₂)_(n)—OH  (L).
 41. The method of claim 39, wherein n isfrom 7 to
 13. 42. The method of claim 40, wherein n is from 7 to
 13. 43.The method of claim 39, wherein Ar₁, Ar₂, and Ar₃ are phenyl.
 44. Themethod of claim 39, wherein X is CH₂O.
 45. The method of claim 39,wherein Q₁ is a C₁-C₆ alkylenyl, optionally having a substituentselected from the group consisting of amino, C₁-C₆ alkylamino, C₁-C₆haloalkylamino, C₁-C₆ haloalkyl C₁-C₆ alkyl amino, C₁-C₆hydroxyalkylamino, C₁-C₆ hydroxyalkyl C₁-C₆ alkylamino, C₁-C₆dialkylamino, C₁-C₆ trialkylamino, and a heterocyclic containing anitrogen atom which may be optionally quaternized.
 46. A method ofkilling a prokaryote comprising contacting the prokaryote with aneffective amount of the compound of claim 1 to reduce or eliminate theproduction of NAD.
 47. A method of killing a prokaryote comprisingcontacting the prokaryote with an effective amount of the compound ofclaim 24 to reduce or eliminate the production of NAD.
 48. A method ofdecreasing prokaryotic growth, comprising contacting the prokaryote withan effective amount of a compound of claim 1 to reduce or eliminate theproduction of NAD.
 49. A method of decreasing prokaryotic growth,comprising contacting the prokaryote with an effective amount of acompound of claim 24 to reduce or eliminate the production of NAD. 50.The method of claim 46, wherein the prokaryote is a bacterium.
 51. Themethod of claim 47, wherein the prokaryote is a bacterium.
 52. Themethod of claim 48, wherein the prokaryote is a bacterium.
 53. Themethod of claim 50, wherein the bacterium is a gram negative or a grampositive bacterium.
 54. The method of claim 51, wherein the bacterium isa gram negative or a gram positive bacterium.
 55. The method of claim52, wherein the bacterium is a gram negative or a gram positivebacterium.
 56. The method of claim 50, wherein the prokaryote is anantibiotic resistant strain of a bacterium.
 57. The method of claim 51,wherein the prokaryote is an antibiotic resistant strain of a bacterium.58. The method of claim 52, wherein the prokaryote is an antibioticresistant strain of a bacterium.
 59. A disinfecting, sterilizing, ordecontaminating composition comprising a compound of claim
 1. 60. Adisinfecting, sterilizing, or decontaminating composition comprising acompound of claim
 24. 61. A method of disinfecting, sterilizing, ordecontaminating a material in need thereof, comprising contacting thematerial with a compound of claim
 1. 62. A method of disinfecting,sterilizing, or decontaminating a material in need thereof, comprisingcontacting the material with a compound of claim
 24. 63. A method ofkilling a fungus comprising contacting the fungus with an amount of acompound of claim 1 to reduce or eliminate the production of NAD.
 64. Amethod of killing a fungus comprising contacting the fungus with anamount of a compound of claim 24 to reduce or eliminate the productionof NAD.
 65. A method of decreasing fungus growth comprising contactingthe fungus with an effective amount of a compound of claim 1 to reduceor eliminate the production of NAD.
 66. A method of decreasing fungusgrowth comprising contacting the fungus with an effective amount of acompound of claim 24 to reduce or eliminate the production of NAD.
 67. Amethod of increasing production of a food animal comprisingadministering to the food animal an effective amount of a compound ofclaim 1 to inhibit the NAD synthetase of a microbe capable of infectingthe food animal.
 68. A method of increasing production of a food animalcomprising administering to the food animal an effective amount of acompound of claim 24 to inhibit the NAD synthetase of a microbe capableof infecting the food animal.
 69. A method for the treatment orprevention of infection by a spore-forming bacterium in an animalcomprising contacting an environment of the animal with an effectiveamount of a compound of claim 1 to inhibit the NAD synthetase of thespore-forming bacterium.
 70. A method for the treatment or prevention ofinfection by a spore-forming bacterium in an animal comprisingcontacting an environment of the animal with an effective amount of acompound of claim 24 to inhibit the NAD synthetase of the spore-formingbacterium.
 71. A method of killing the vegetative cell of aspore-forming bacterium in an environment comprising treating theenvironment with an effective amount of a compound of claim 1 to inhibitthe NAD synthetase of the bacterium.
 72. A method of killing thevegetative cell of a spore-forming bacterium in an environmentcomprising treating the environment with an effective amount of acompound of claim 24 to inhibit the NAD synthetase of the bacterium. 73.A method of treating or preventing a microbial infection or disease in aplant comprising contacting the plant or an environment of the plantwith an effective amount of a compound of claim 1 to inhibit the NADsynthetase of the microbe.
 74. A method of treating or preventing amicrobial infection or disease in a plant comprising contacting theplant or an environment of the plant with an effective amount of acompound of claim 24 to inhibit the NAD synthetase of the microbe.
 75. Amethod for a treating or preventing harm to a plant due to a pestcomprising contacting the plant, or an environment thereof, with apesticidal effective amount of a compound of claim 1 to inhibit the NADsynthetase of a pest.
 76. A method for a treating or preventing harm toa plant due to a pest comprising contacting the plant, or an environmentthereof, with a pesticidal effective amount of a compound of claim 24 toinhibit the NAD synthetase of a pest.
 77. A method of controlling insectpopulation in an environment comprising contacting the environment withan effective amount of a compound of claim 1 to inhibit the NADsynthetase of the insect.
 78. A method of controlling insect populationin an environment comprising contacting the environment with aneffective amount of a compound of claim 24 to inhibit the NAD synthetaseof the insect.
 79. A method for combating agroterrorism involving aninfective agent on an object comprising treating the object with anamount of a compound effective to inhibit the NAD synthetase of theinfective agent.
 80. The method of claim 79, wherein the object is ananimal, crop, or soil.
 81. The method of claim 79, wherein the infectiveagent is a fungus.
 82. The method of claim 79, wherein the infectiveagent is a bacterium.
 83. The method of claim 79, wherein the compoundis a compound of claim
 1. 84. The method of claim 79, wherein thecompound is a compound of claim
 24. 85. The compound of claim 22, whichis selected from the group consisting of:

wherein I⁻ is a pharmaceutically acceptable anion.